<?xml version="1.0" encoding="US-ASCII" standalone="no"?>
<!--Generated by ThunderDome XBRL - 06:45PM UTC 2026-08-05--><link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" xlink:type="simple"/>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-7-net-loss-per-common-share" xlink:href="phio-20260630.xsd#statement-note-7-net-loss-per-common-share" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-7-net-loss-per-common-share" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareTextBlock" xlink:label="us-gaap_EarningsPerShareTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_EarningsPerShareTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-2-accounts-payable-and-accrued-liabilities" xlink:href="phio-20260630.xsd#statement-note-2-accounts-payable-and-accrued-liabilities" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-2-accounts-payable-and-accrued-liabilities" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsPayableAndAccruedLiabilitiesDisclosureTextBlock" xlink:label="us-gaap_AccountsPayableAndAccruedLiabilitiesDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OffsettingAssetsTable" xlink:label="us-gaap_OffsettingAssetsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_OffsettingAssetsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OffsettingAssetsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_AccountsPayableAndAccruedLiabilitiesDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-document-and-entity-information" xlink:href="phio-20260630.xsd#statement-document-and-entity-information" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-document-and-entity-information" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_AmendmentFlag" xlink:label="dei_AmendmentFlag" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_CityAreaCode" xlink:label="dei_CityAreaCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_CurrentFiscalYearEndDate" xlink:label="dei_CurrentFiscalYearEndDate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentFiscalPeriodFocus" xlink:label="dei_DocumentFiscalPeriodFocus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentFiscalYearFocus" xlink:label="dei_DocumentFiscalYearFocus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentInformationLineItems" xlink:label="dei_DocumentInformationLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentInformationTable" xlink:label="dei_DocumentInformationTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentPeriodEndDate" xlink:label="dei_DocumentPeriodEndDate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentQuarterlyReport" xlink:label="dei_DocumentQuarterlyReport" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentTransitionReport" xlink:label="dei_DocumentTransitionReport" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_DocumentType" xlink:label="dei_DocumentType" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressAddressLine1" xlink:label="dei_EntityAddressAddressLine1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressCityOrTown" xlink:label="dei_EntityAddressCityOrTown" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressPostalZipCode" xlink:label="dei_EntityAddressPostalZipCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityAddressStateOrProvince" xlink:label="dei_EntityAddressStateOrProvince" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCentralIndexKey" xlink:label="dei_EntityCentralIndexKey" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCommonStockSharesOutstanding" xlink:label="dei_EntityCommonStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityCurrentReportingStatus" xlink:label="dei_EntityCurrentReportingStatus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityEmergingGrowthCompany" xlink:label="dei_EntityEmergingGrowthCompany" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityFileNumber" xlink:label="dei_EntityFileNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityFilerCategory" xlink:label="dei_EntityFilerCategory" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityIncorporationStateCountryCode" xlink:label="dei_EntityIncorporationStateCountryCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityInteractiveDataCurrent" xlink:label="dei_EntityInteractiveDataCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityRegistrantName" xlink:label="dei_EntityRegistrantName" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityShellCompany" xlink:label="dei_EntityShellCompany" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntitySmallBusiness" xlink:label="dei_EntitySmallBusiness" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityTaxIdentificationNumber" xlink:label="dei_EntityTaxIdentificationNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LocalPhoneNumber" xlink:label="dei_LocalPhoneNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_Security12bTitle" xlink:label="dei_Security12bTitle" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_SecurityExchangeName" xlink:label="dei_SecurityExchangeName" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_TradingSymbol" xlink:label="dei_TradingSymbol" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="dei_DocumentInformationTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentInformationTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCentralIndexKey" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityRegistrantName" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AmendmentFlag" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CurrentFiscalYearEndDate" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalPeriodFocus" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalYearFocus" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentType" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentQuarterlyReport" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentPeriodEndDate" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentTransitionReport" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFileNumber" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityIncorporationStateCountryCode" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityTaxIdentificationNumber" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressAddressLine1" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressCityOrTown" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressStateOrProvince" xlink:type="arc"/>
    <link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressPostalZipCode" xlink:type="arc"/>
    <link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CityAreaCode" xlink:type="arc"/>
    <link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_LocalPhoneNumber" xlink:type="arc"/>
    <link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_Security12bTitle" xlink:type="arc"/>
    <link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_TradingSymbol" xlink:type="arc"/>
    <link:definitionArc order="22" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_SecurityExchangeName" xlink:type="arc"/>
    <link:definitionArc order="23" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCurrentReportingStatus" xlink:type="arc"/>
    <link:definitionArc order="24" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityInteractiveDataCurrent" xlink:type="arc"/>
    <link:definitionArc order="25" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFilerCategory" xlink:type="arc"/>
    <link:definitionArc order="26" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntitySmallBusiness" xlink:type="arc"/>
    <link:definitionArc order="27" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityEmergingGrowthCompany" xlink:type="arc"/>
    <link:definitionArc order="28" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityShellCompany" xlink:type="arc"/>
    <link:definitionArc order="29" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCommonStockSharesOutstanding" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-condensed-consolidated-balance-sheets-current-period-unaudited-parentheticals" xlink:href="phio-20260630.xsd#statement-condensed-consolidated-balance-sheets-current-period-unaudited-parentheticals" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-condensed-consolidated-balance-sheets-current-period-unaudited-parentheticals" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="us-gaap_PreferredStockSharesIssued" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="us-gaap_PreferredStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesIssued" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesIssued" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesOutstanding" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-1-organization-and-significant-accounting-policies" xlink:href="phio-20260630.xsd#statement-note-1-organization-and-significant-accounting-policies" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-1-organization-and-significant-accounting-policies" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:label="us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OrganizationConsolidationBasisOfPresentationBusinessDescriptionAndAccountingPoliciesTextBlock" xlink:label="us-gaap_OrganizationConsolidationBasisOfPresentationBusinessDescriptionAndAccountingPoliciesTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_OrganizationConsolidationBasisOfPresentationBusinessDescriptionAndAccountingPoliciesTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-3-collaboration-agreement" xlink:href="phio-20260630.xsd#statement-note-3-collaboration-agreement" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-3-collaboration-agreement" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_AgonoxMember" xlink:label="phio_AgonoxMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_CollaborativeArrangementClinicalCodevelopmentAgreementMember" xlink:label="phio_CollaborativeArrangementClinicalCodevelopmentAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CollaborativeArrangementDisclosureTextBlock" xlink:label="us-gaap_CollaborativeArrangementDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="phio_CollaborativeArrangementClinicalCodevelopmentAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="phio_AgonoxMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_CollaborativeArrangementDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-4-leases" xlink:href="phio-20260630.xsd#statement-note-4-leases" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-4-leases" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_FormerCorporateHeadquartersMember" xlink:label="phio_FormerCorporateHeadquartersMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_LaboratoryFacilityWorcesterMassachusettsMember" xlink:label="phio_LaboratoryFacilityWorcesterMassachusettsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LeaseContractualTermAxis" xlink:label="us-gaap_LeaseContractualTermAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LesseeLeaseDescriptionTable" xlink:label="us-gaap_LesseeLeaseDescriptionTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LesseeOperatingLeasesTextBlock" xlink:label="us-gaap_LesseeOperatingLeasesTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_LesseeLeaseDescriptionTable" xlink:to="us-gaap_LeaseContractualTermAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LeaseContractualTermDomain" xlink:to="phio_LaboratoryFacilityWorcesterMassachusettsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LeaseContractualTermDomain" xlink:to="phio_FormerCorporateHeadquartersMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_LesseeLeaseDescriptionTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeLeaseDescriptionTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_LesseeOperatingLeasesTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity" xlink:href="phio-20260630.xsd#statement-note-5-stockholders-equity" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_AtTheMarketAgreementMember" xlink:label="phio_AtTheMarketAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_CommonStockWarrantsMember" xlink:label="phio_CommonStockWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarantsMember" xlink:label="phio_ExistingWarantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarrantsDecember2024Member" xlink:label="phio_ExistingWarrantsDecember2024Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarrantsJanuary2025Member" xlink:label="phio_ExistingWarrantsJanuary2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarrantsJuly2025Member" xlink:label="phio_ExistingWarrantsJuly2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_HcWainwrightCoLlcMember" xlink:label="phio_HcWainwrightCoLlcMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_January132025OfferingsMember" xlink:label="phio_January132025OfferingsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_January142025OfferingsMember" xlink:label="phio_January142025OfferingsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_January162025OfferingsMember" xlink:label="phio_January162025OfferingsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_July2025FinancingMember" xlink:label="phio_July2025FinancingMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_NewWarrantsJuly2025Member" xlink:label="phio_NewWarrantsJuly2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_NewWarrantsNovember2025Member" xlink:label="phio_NewWarrantsNovember2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_November2025FinancingMember" xlink:label="phio_November2025FinancingMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsMember" xlink:label="phio_PlacementAgentWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsOneMember" xlink:label="phio_PlacementAgentWarrantsOneMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsThreeMember" xlink:label="phio_PlacementAgentWarrantsThreeMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsTwoMember" xlink:label="phio_PlacementAgentWarrantsTwoMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_RegisteredDirectOfferingMember" xlink:label="phio_RegisteredDirectOfferingMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesAWarrantsMember" xlink:label="phio_SeriesAWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesGWarrantsMember" xlink:label="phio_SeriesGWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesHWarrantsMember" xlink:label="phio_SeriesHWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesIWarrantsMember" xlink:label="phio_SeriesIWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesJWarrantsMember" xlink:label="phio_SeriesJWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesKWarrantsMember" xlink:label="phio_SeriesKWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCapitalizationEquityTable" xlink:label="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:label="us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="phio_HcWainwrightCoLlcMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_January132025OfferingsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesGWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_January142025OfferingsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_RegisteredDirectOfferingMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesHWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_CommonStockWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_January162025OfferingsMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesIWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarrantsDecember2024Member" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarrantsJanuary2025Member" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_NewWarrantsJuly2025Member" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesJWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesKWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsOneMember" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsTwoMember" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsThreeMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_July2025FinancingMember" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarrantsJuly2025Member" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_NewWarrantsNovember2025Member" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesAWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_November2025FinancingMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_AtTheMarketAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation" xlink:href="phio-20260630.xsd#statement-note-6-stockbased-compensation" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_The2020PlanMember" xlink:label="phio_The2020PlanMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:label="us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusAxis" xlink:label="us-gaap_GranteeStatusAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:label="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="phio_The2020PlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_GranteeStatusAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-8-segment-information" xlink:href="phio-20260630.xsd#statement-note-8-segment-information" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-8-segment-information" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingDisclosureTextBlock" xlink:label="us-gaap_SegmentReportingDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_SegmentReportingDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-significant-accounting-policies-policies" xlink:href="phio-20260630.xsd#statement-significant-accounting-policies-policies" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-significant-accounting-policies-policies" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_LiquidityPolicyTextBlock" xlink:label="phio_LiquidityPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PolicyTable" xlink:label="phio_PolicyTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountingPoliciesAbstract" xlink:label="us-gaap_AccountingPoliciesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_BasisOfAccountingPolicyPolicyTextBlock" xlink:label="us-gaap_BasisOfAccountingPolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ConsolidationPolicyTextBlock" xlink:label="us-gaap_ConsolidationPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeTaxPolicyTextBlock" xlink:label="us-gaap_IncomeTaxPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:label="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PolicyTextBlockAbstract" xlink:label="us-gaap_PolicyTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingPolicyPolicyTextBlock" xlink:label="us-gaap_SegmentReportingPolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_UseOfEstimates" xlink:label="us-gaap_UseOfEstimates" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PolicyTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="phio_PolicyTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_PolicyTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PolicyTextBlockAbstract" xlink:to="us-gaap_AccountingPoliciesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_BasisOfAccountingPolicyPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ConsolidationPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_SegmentReportingPolicyPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_UseOfEstimates" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="phio_LiquidityPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_IncomeTaxPolicyTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-2-accounts-payable-and-accrued-liabilities-tables" xlink:href="phio-20260630.xsd#statement-note-2-accounts-payable-and-accrued-liabilities-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-2-accounts-payable-and-accrued-liabilities-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OffsettingAssetsTable" xlink:label="us-gaap_OffsettingAssetsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAccountsPayableAndAccruedLiabilitiesTableTextBlock" xlink:label="us-gaap_ScheduleOfAccountsPayableAndAccruedLiabilitiesTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_OffsettingAssetsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OffsettingAssetsTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAccountsPayableAndAccruedLiabilitiesTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity-tables" xlink:href="phio-20260630.xsd#statement-note-5-stockholders-equity-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCapitalizationEquityTable" xlink:label="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:label="us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-tables" xlink:href="phio-20260630.xsd#statement-note-6-stockbased-compensation-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfNonvestedRestrictedStockUnitsActivityTableTextBlock" xlink:label="us-gaap_ScheduleOfNonvestedRestrictedStockUnitsActivityTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfNonvestedRestrictedStockUnitsActivityTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-7-net-loss-per-common-share-tables" xlink:href="phio-20260630.xsd#statement-note-7-net-loss-per-common-share-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-7-net-loss-per-common-share-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-8-segment-information-tables" xlink:href="phio-20260630.xsd#statement-note-8-segment-information-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-8-segment-information-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTextBlock" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-1-organization-and-significant-accounting-policies-details-textual" xlink:href="phio-20260630.xsd#statement-note-1-organization-and-significant-accounting-policies-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-1-organization-and-significant-accounting-policies-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:label="us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NumberOfOperatingSegments" xlink:label="us-gaap_NumberOfOperatingSegments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NewAccountingPronouncementsOrChangeInAccountingPrincipleTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NumberOfOperatingSegments" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-3-collaboration-agreement-details-textual" xlink:href="phio-20260630.xsd#statement-note-3-collaboration-agreement-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-3-collaboration-agreement-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_AgonoxMember" xlink:label="phio_AgonoxMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_CollaborativeArrangementClinicalCodevelopmentAgreementMember" xlink:label="phio_CollaborativeArrangementClinicalCodevelopmentAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PaymentsForAccruedObligationsOnCollaborativeArrangements" xlink:label="phio_PaymentsForAccruedObligationsOnCollaborativeArrangements" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractualObligation" xlink:label="us-gaap_ContractualObligation" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="phio_CollaborativeArrangementClinicalCodevelopmentAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="phio_AgonoxMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ContractualObligation" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_PaymentsForAccruedObligationsOnCollaborativeArrangements" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-4-leases-details-textual" xlink:href="phio-20260630.xsd#statement-note-4-leases-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-4-leases-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_FormerCorporateHeadquartersMember" xlink:label="phio_FormerCorporateHeadquartersMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_LaboratoryFacilityWorcesterMassachusettsMember" xlink:label="phio_LaboratoryFacilityWorcesterMassachusettsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_OperatingLeasesMonthlyPayment" xlink:label="phio_OperatingLeasesMonthlyPayment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LeaseContractualTermAxis" xlink:label="us-gaap_LeaseContractualTermAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LesseeLeaseDescriptionTable" xlink:label="us-gaap_LesseeLeaseDescriptionTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingLeaseExpense" xlink:label="us-gaap_OperatingLeaseExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingLeasePayments" xlink:label="us-gaap_OperatingLeasePayments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_LesseeLeaseDescriptionTable" xlink:to="us-gaap_LeaseContractualTermAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LeaseContractualTermDomain" xlink:to="phio_LaboratoryFacilityWorcesterMassachusettsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LeaseContractualTermDomain" xlink:to="phio_FormerCorporateHeadquartersMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_LesseeLeaseDescriptionTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeLeaseDescriptionTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_OperatingLeasesMonthlyPayment" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseExpense" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeasePayments" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity-details-textual" xlink:href="phio-20260630.xsd#statement-note-5-stockholders-equity-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_AtTheMarketAgreementMember" xlink:label="phio_AtTheMarketAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightExercisedDuringPeriod" xlink:label="phio_ClassOfWarrantOrRightExercisedDuringPeriod" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightExercisedDuringPeriodAtAReducedPrice" xlink:label="phio_ClassOfWarrantOrRightExercisedDuringPeriodAtAReducedPrice" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightExercisedDuringPeriodExercisePrice" xlink:label="phio_ClassOfWarrantOrRightExercisedDuringPeriodExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightPurchasePriceOfWarrantsOrRights" xlink:label="phio_ClassOfWarrantOrRightPurchasePriceOfWarrantsOrRights" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_CommonStockWarrantsMember" xlink:label="phio_CommonStockWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_EquityIssuableMaximumValue" xlink:label="phio_EquityIssuableMaximumValue" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarantsMember" xlink:label="phio_ExistingWarantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarrantsDecember2024Member" xlink:label="phio_ExistingWarrantsDecember2024Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarrantsJanuary2025Member" xlink:label="phio_ExistingWarrantsJanuary2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ExistingWarrantsJuly2025Member" xlink:label="phio_ExistingWarrantsJuly2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_HcWainwrightCoLlcMember" xlink:label="phio_HcWainwrightCoLlcMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_January132025OfferingsMember" xlink:label="phio_January132025OfferingsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_January142025OfferingsMember" xlink:label="phio_January142025OfferingsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_January162025OfferingsMember" xlink:label="phio_January162025OfferingsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_July2025FinancingMember" xlink:label="phio_July2025FinancingMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_NewWarrantsJuly2025Member" xlink:label="phio_NewWarrantsJuly2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_NewWarrantsNovember2025Member" xlink:label="phio_NewWarrantsNovember2025Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_November2025FinancingMember" xlink:label="phio_November2025FinancingMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_NumberOfWarrantsPerUnit" xlink:label="phio_NumberOfWarrantsPerUnit" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsMember" xlink:label="phio_PlacementAgentWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsOneMember" xlink:label="phio_PlacementAgentWarrantsOneMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsThreeMember" xlink:label="phio_PlacementAgentWarrantsThreeMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_PlacementAgentWarrantsTwoMember" xlink:label="phio_PlacementAgentWarrantsTwoMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_RegisteredDirectOfferingMember" xlink:label="phio_RegisteredDirectOfferingMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesAWarrantsMember" xlink:label="phio_SeriesAWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesGWarrantsMember" xlink:label="phio_SeriesGWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesHWarrantsMember" xlink:label="phio_SeriesHWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesIWarrantsMember" xlink:label="phio_SeriesIWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesJWarrantsMember" xlink:label="phio_SeriesJWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_SeriesKWarrantsMember" xlink:label="phio_SeriesKWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight" xlink:label="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:label="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:label="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromWarrantExercises" xlink:label="us-gaap_ProceedsFromWarrantExercises" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCapitalizationEquityTable" xlink:label="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="us-gaap_SharesIssuedPricePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstanding" xlink:label="us-gaap_WarrantsAndRightsOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstandingTerm" xlink:label="us-gaap_WarrantsAndRightsOutstandingTerm" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="phio_HcWainwrightCoLlcMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_January132025OfferingsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesGWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_January142025OfferingsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_RegisteredDirectOfferingMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesHWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_CommonStockWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_January162025OfferingsMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesIWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarrantsDecember2024Member" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarrantsJanuary2025Member" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_NewWarrantsJuly2025Member" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesJWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesKWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsOneMember" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsTwoMember" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_PlacementAgentWarrantsThreeMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_July2025FinancingMember" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_ExistingWarrantsJuly2025Member" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_NewWarrantsNovember2025Member" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="phio_SeriesAWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_November2025FinancingMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="phio_AtTheMarketAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesIssuedPricePerShare" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_NumberOfWarrantsPerUnit" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightExercisedDuringPeriod" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightExercisedDuringPeriodExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightExercisedDuringPeriodAtAReducedPrice" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightPurchasePriceOfWarrantsOrRights" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WarrantsAndRightsOutstanding" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WarrantsAndRightsOutstandingTerm" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromWarrantExercises" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_EquityIssuableMaximumValue" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-details-textual" xlink:href="phio-20260630.xsd#statement-note-6-stockbased-compensation-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_The2020PlanMember" xlink:label="phio_The2020PlanMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusAxis" xlink:label="us-gaap_GranteeStatusAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:label="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="phio_The2020PlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_GranteeStatusAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-8-segment-information-details-textual" xlink:href="phio-20260630.xsd#statement-note-8-segment-information-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-8-segment-information-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NumberOfOperatingSegments" xlink:label="us-gaap_NumberOfOperatingSegments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NumberOfReportableSegments" xlink:label="us-gaap_NumberOfReportableSegments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NumberOfOperatingSegments" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NumberOfReportableSegments" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-2-accounts-payable-and-accrued-liabilities-schedule-of-accrued-expenses-details" xlink:href="phio-20260630.xsd#statement-note-2-accounts-payable-and-accrued-liabilities-schedule-of-accrued-expenses-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-2-accounts-payable-and-accrued-liabilities-schedule-of-accrued-expenses-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ResearchAndDevelopmentAccruals" xlink:label="phio_ResearchAndDevelopmentAccruals" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsPayableAndAccruedLiabilitiesCurrentAndNoncurrent" xlink:label="us-gaap_AccountsPayableAndAccruedLiabilitiesCurrentAndNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsPayableTradeCurrentAndNoncurrent" xlink:label="us-gaap_AccountsPayableTradeCurrentAndNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccruedProfessionalFeesCurrent" xlink:label="us-gaap_AccruedProfessionalFeesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeRelatedLiabilitiesCurrentAndNoncurrent" xlink:label="us-gaap_EmployeeRelatedLiabilitiesCurrentAndNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OffsettingAssetsTable" xlink:label="us-gaap_OffsettingAssetsTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_OffsettingAssetsTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OffsettingAssetsTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccountsPayableTradeCurrentAndNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ResearchAndDevelopmentAccruals" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeRelatedLiabilitiesCurrentAndNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccruedProfessionalFeesCurrent" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccountsPayableAndAccruedLiabilitiesCurrentAndNoncurrent" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-7-net-loss-per-common-share-schedule-of-antidilutive-securities-details" xlink:href="phio-20260630.xsd#statement-note-7-net-loss-per-common-share-schedule-of-antidilutive-securities-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-7-net-loss-per-common-share-schedule-of-antidilutive-securities-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_WarrantMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-condensed-consolidated-balance-sheets-current-period-unaudited" xlink:href="phio-20260630.xsd#statement-condensed-consolidated-balance-sheets-current-period-unaudited" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-condensed-consolidated-balance-sheets-current-period-unaudited" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccountsPayableCurrent" xlink:label="us-gaap_AccountsPayableCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapital" xlink:label="us-gaap_AdditionalPaidInCapital" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Assets" xlink:label="us-gaap_Assets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsAbstract" xlink:label="us-gaap_AssetsAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrent" xlink:label="us-gaap_AssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsCurrentAbstract" xlink:label="us-gaap_AssetsCurrentAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommitmentsAndContingencies" xlink:label="us-gaap_CommitmentsAndContingencies" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockValue" xlink:label="us-gaap_CommonStockValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Liabilities" xlink:label="us-gaap_Liabilities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:label="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:label="us-gaap_LiabilitiesCurrentAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockValue" xlink:label="us-gaap_PreferredStockValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsCurrentAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AssetsCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_Assets" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesCurrentAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccountsPayableCurrent" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccruedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_Liabilities" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_CommitmentsAndContingencies" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_PreferredStockValue" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_CommonStockValue" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_AdditionalPaidInCapital" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-condensed-consolidated-statements-of-operations-unaudited" xlink:href="phio-20260630.xsd#statement-condensed-consolidated-statements-of-operations-unaudited" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-condensed-consolidated-statements-of-operations-unaudited" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareAbstract" xlink:label="us-gaap_EarningsPerShareAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareBasic" xlink:label="us-gaap_EarningsPerShareBasic" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EarningsPerShareBasicOtherDisclosuresAbstract" xlink:label="us-gaap_EarningsPerShareBasicOtherDisclosuresAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InvestmentIncomeNet" xlink:label="us-gaap_InvestmentIncomeNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingExpenses" xlink:label="us-gaap_OperatingExpenses" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingExpensesAbstract" xlink:label="us-gaap_OperatingExpensesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingIncomeLoss" xlink:label="us-gaap_OperatingIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherNonoperatingIncomeExpense" xlink:label="us-gaap_OtherNonoperatingIncomeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:label="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingExpensesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_GeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_OperatingExpenses" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_OperatingIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_InvestmentIncomeNet" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_OtherNonoperatingIncomeExpense" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EarningsPerShareAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareAbstract" xlink:to="us-gaap_EarningsPerShareBasic" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EarningsPerShareBasicOtherDisclosuresAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EarningsPerShareBasicOtherDisclosuresAbstract" xlink:to="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-condensed-consolidated-statements-of-cash-flows-unaudited" xlink:href="phio-20260630.xsd#statement-condensed-consolidated-statements-of-cash-flows-unaudited" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-condensed-consolidated-statements-of-cash-flows-unaudited" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ProceedsFromIssuanceOfCommonStockAndWarrants" xlink:label="phio_ProceedsFromIssuanceOfCommonStockAndWarrants" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DepreciationDepletionAndAmortization" xlink:label="us-gaap_DepreciationDepletionAndAmortization" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GainLossOnSaleOfPropertyPlantEquipment" xlink:label="us-gaap_GainLossOnSaleOfPropertyPlantEquipment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInAccountsPayable" xlink:label="us-gaap_IncreaseDecreaseInAccountsPayable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInAccruedLiabilities" xlink:label="us-gaap_IncreaseDecreaseInAccruedLiabilities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:label="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:label="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:label="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromSaleOfProductiveAssets" xlink:label="us-gaap_ProceedsFromSaleOfProductiveAssets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromWarrantExercises" xlink:label="us-gaap_ProceedsFromWarrantExercises" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensation" xlink:label="us-gaap_ShareBasedCompensation" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_DepreciationDepletionAndAmortization" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_GainLossOnSaleOfPropertyPlantEquipment" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ShareBasedCompensation" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsPayable" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccruedLiabilities" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromSaleOfProductiveAssets" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromWarrantExercises" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="phio_ProceedsFromIssuanceOfCommonStockAndWarrants" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-condensed-consolidated-statements-of-stockholders-equity-unaudited" xlink:href="phio-20260630.xsd#statement-condensed-consolidated-statements-of-stockholders-equity-unaudited" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-condensed-consolidated-statements-of-stockholders-equity-unaudited" xlink:type="extended">
    <link:loc xlink:href="phio-20260630.xsd#phio_CommonStockOutstandingMember" xlink:label="phio_CommonStockOutstandingMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_StockIssuedDuringPeriodSharesStocksAndWarrants" xlink:label="phio_StockIssuedDuringPeriodSharesStocksAndWarrants" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_StockIssuedDuringPeriodSharesWarrantsExercised" xlink:label="phio_StockIssuedDuringPeriodSharesWarrantsExercised" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_StockIssuedDuringPeriodValueStocksAndWarrants" xlink:label="phio_StockIssuedDuringPeriodValueStocksAndWarrants" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_StockIssuedDuringPeriodValueWarrantsExercised" xlink:label="phio_StockIssuedDuringPeriodValueWarrantsExercised" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesOutstanding" xlink:label="us-gaap_SharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="phio_CommonStockOutstandingMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_StockIssuedDuringPeriodSharesWarrantsExercised" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_StockIssuedDuringPeriodValueWarrantsExercised" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_StockIssuedDuringPeriodSharesStocksAndWarrants" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_StockIssuedDuringPeriodValueStocksAndWarrants" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-total-stockbased-compensation-expense-details" xlink:href="phio-20260630.xsd#statement-note-6-stockbased-compensation-total-stockbased-compensation-expense-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-total-stockbased-compensation-expense-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_GeneralAndAdministrativeMember" xlink:label="phio_GeneralAndAdministrativeMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ResearchAndDevelopmentMember" xlink:label="phio_ResearchAndDevelopmentMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:label="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:label="us-gaap_InterimPeriodCostsNotAllocableDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NatureOfExpenseAxis" xlink:label="us-gaap_NatureOfExpenseAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_NatureOfExpenseAxis" xlink:to="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_NatureOfExpenseAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:to="phio_ResearchAndDevelopmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_NatureOfExpenseAxis" xlink:to="us-gaap_InterimPeriodCostsNotAllocableDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:to="phio_GeneralAndAdministrativeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-8-segment-information-schedule-of-segment-reporting-information-details" xlink:href="phio-20260630.xsd#statement-note-8-segment-information-schedule-of-segment-reporting-information-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-8-segment-information-schedule-of-segment-reporting-information-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_EmployeeRelatedResearchExpensesMember" xlink:label="phio_EmployeeRelatedResearchExpensesMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ReportableSegmentMember" xlink:label="phio_ReportableSegmentMember" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ResearchOfPh762Member" xlink:label="phio_ResearchOfPh762Member" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ResearchOfPh894Member" xlink:label="phio_ResearchOfPh894Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsAxis" xlink:label="srt_ConsolidationItemsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain" xlink:label="srt_ConsolidationItemsDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ConsolidationItemsDomain" xlink:label="srt_ConsolidationItemsDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:label="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:label="us-gaap_InterimPeriodCostsNotAllocableDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NatureOfExpenseAxis" xlink:label="us-gaap_NatureOfExpenseAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingExpenses" xlink:label="us-gaap_OperatingExpenses" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingSegmentsMember" xlink:label="us-gaap_OperatingSegmentsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherNonoperatingIncome" xlink:label="us-gaap_OtherNonoperatingIncome" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain" xlink:label="us-gaap_SegmentDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:label="us-gaap_StatementBusinessSegmentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ConsolidationItemsAxis" xlink:to="srt_ConsolidationItemsDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_NatureOfExpenseAxis" xlink:to="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="srt_ConsolidationItemsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ConsolidationItemsDomain" xlink:to="us-gaap_OperatingSegmentsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ConsolidationItemsAxis" xlink:to="srt_ConsolidationItemsDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_NatureOfExpenseAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:to="phio_ResearchOfPh762Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_NatureOfExpenseAxis" xlink:to="us-gaap_InterimPeriodCostsNotAllocableDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentDomain" xlink:to="phio_ReportableSegmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:to="phio_ResearchOfPh894Member" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InterimPeriodCostsNotAllocableDomain" xlink:to="phio_EmployeeRelatedResearchExpensesMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingExpenses" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherNonoperatingIncome" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity-schedule-of-outstanding-warrants-details" xlink:href="phio-20260630.xsd#statement-note-5-stockholders-equity-schedule-of-outstanding-warrants-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-5-stockholders-equity-schedule-of-outstanding-warrants-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightExercisedDuringPeriod" xlink:label="phio_ClassOfWarrantOrRightExercisedDuringPeriod" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightExercisedDuringPeriodExercisePrice" xlink:label="phio_ClassOfWarrantOrRightExercisedDuringPeriodExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightExpiredDuringPeriod" xlink:label="phio_ClassOfWarrantOrRightExpiredDuringPeriod" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightExpiredDuringPeriodExercisePrice" xlink:label="phio_ClassOfWarrantOrRightExpiredDuringPeriodExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightIssuedDuringPeriod" xlink:label="phio_ClassOfWarrantOrRightIssuedDuringPeriod" xlink:type="locator"/>
    <link:loc xlink:href="phio-20260630.xsd#phio_ClassOfWarrantOrRightIssuedDuringPeriodExercisePrice" xlink:label="phio_ClassOfWarrantOrRightIssuedDuringPeriodExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCapitalizationEquityTable" xlink:label="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfCapitalizationEquityTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightIssuedDuringPeriod" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightIssuedDuringPeriodExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightExercisedDuringPeriod" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightExercisedDuringPeriodExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightExpiredDuringPeriod" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="phio_ClassOfWarrantOrRightExpiredDuringPeriodExercisePrice" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-restricted-stock-units-activity-details" xlink:href="phio-20260630.xsd#statement-note-6-stockbased-compensation-restricted-stock-units-activity-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://phiopharma.com/20260630/role/statement-note-6-stockbased-compensation-restricted-stock-units-activity-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:type="arc"/>
  </link:definitionLink>
</link:linkbase>
