<?xml version="1.0" encoding="US-ASCII" standalone="no"?>
    <!-- Field: Doc-Info; Name: Generator; Value: GoFiler Complete; Version: 6.5a -->
    <!-- Field: Doc-Info; Name: VendorURI; Value: https://www.novaworks.com -->
    <!-- Field: Doc-Info; Name: Status; Value: 0x00000000 -->
<link:linkbase xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xbrli="http://www.xbrl.org/2003/instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#Cover" roleURI="http://pulmatrix.com/role/Cover" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#BalanceSheets" roleURI="http://pulmatrix.com/role/BalanceSheets" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#BalanceSheetsParenthetical" roleURI="http://pulmatrix.com/role/BalanceSheetsParenthetical" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#StatementsOfOperations" roleURI="http://pulmatrix.com/role/StatementsOfOperations" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#StatementsOfStockholdersEquity" roleURI="http://pulmatrix.com/role/StatementsOfStockholdersEquity" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#StatementsOfCashFlows" roleURI="http://pulmatrix.com/role/StatementsOfCashFlows" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#Organization" roleURI="http://pulmatrix.com/role/Organization" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandards" roleURI="http://pulmatrix.com/role/SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandards" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#PrepaidExpensesAndOtherCurrentAssets" roleURI="http://pulmatrix.com/role/PrepaidExpensesAndOtherCurrentAssets" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#AccruedExpensesAndOtherCurrentLiabilities" roleURI="http://pulmatrix.com/role/AccruedExpensesAndOtherCurrentLiabilities" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#PreferredStock" roleURI="http://pulmatrix.com/role/PreferredStock" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#CommonStock" roleURI="http://pulmatrix.com/role/CommonStock" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#Warrants" roleURI="http://pulmatrix.com/role/Warrants" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#Stock-basedCompensation" roleURI="http://pulmatrix.com/role/Stock-basedCompensation" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#CommitmentsAndContingencies" roleURI="http://pulmatrix.com/role/CommitmentsAndContingencies" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#IncomeTaxes" roleURI="http://pulmatrix.com/role/IncomeTaxes" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#NetLossPerShare" roleURI="http://pulmatrix.com/role/NetLossPerShare" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#SegmentReporting" roleURI="http://pulmatrix.com/role/SegmentReporting" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#SubsequentEvents" roleURI="http://pulmatrix.com/role/SubsequentEvents" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandardsPolicies" roleURI="http://pulmatrix.com/role/SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandardsPolicies" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#PrepaidExpensesAndOtherCurrentAssetsTables" roleURI="http://pulmatrix.com/role/PrepaidExpensesAndOtherCurrentAssetsTables" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#AccruedExpensesAndOtherCurrentLiabilitiesTables" roleURI="http://pulmatrix.com/role/AccruedExpensesAndOtherCurrentLiabilitiesTables" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#WarrantsTables" roleURI="http://pulmatrix.com/role/WarrantsTables" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#Stock-basedCompensationTables" roleURI="http://pulmatrix.com/role/Stock-basedCompensationTables" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#NetLossPerShareTables" roleURI="http://pulmatrix.com/role/NetLossPerShareTables" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#OrganizationDetailsNarrative" roleURI="http://pulmatrix.com/role/OrganizationDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandardsDetailsNarrative" roleURI="http://pulmatrix.com/role/SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandardsDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#ScheduleOfPrepaidExpensesAndOtherCurrentAssetsDetails" roleURI="http://pulmatrix.com/role/ScheduleOfPrepaidExpensesAndOtherCurrentAssetsDetails" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#ScheduleOfAccruedExpensesAndOtherCurrentLiabilitiesDetails" roleURI="http://pulmatrix.com/role/ScheduleOfAccruedExpensesAndOtherCurrentLiabilitiesDetails" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#PreferredStockDetailsNarrative" roleURI="http://pulmatrix.com/role/PreferredStockDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#CommonStockDetailsNarrative" roleURI="http://pulmatrix.com/role/CommonStockDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#ScheduleOfWarrantsOutstandingAndExercisableDetails" roleURI="http://pulmatrix.com/role/ScheduleOfWarrantsOutstandingAndExercisableDetails" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#WarrantsDetailsNarrative" roleURI="http://pulmatrix.com/role/WarrantsDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#ScheduleOfStockOptionActivityDetails" roleURI="http://pulmatrix.com/role/ScheduleOfStockOptionActivityDetails" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#Stock-basedCompensationDetailsNarrative" roleURI="http://pulmatrix.com/role/Stock-basedCompensationDetailsNarrative" />
    <link:roleRef xlink:type="simple" xlink:href="pulm-20260331.xsd#ScheduleOfComputationOfAnti-dilutiveWeighted-averageSharesOutstandingDetails" roleURI="http://pulmatrix.com/role/ScheduleOfComputationOfAnti-dilutiveWeighted-averageSharesOutstandingDetails" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" arcroleURI="http://xbrl.org/int/dim/arcrole/all" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#notAll" arcroleURI="http://xbrl.org/int/dim/arcrole/notAll" />
    <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/Cover" xlink:title="00000001 - Document - Cover" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/BalanceSheets" xlink:title="00000002 - Statement - Consolidated Balance Sheets" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/BalanceSheetsParenthetical" xlink:title="00000003 - Statement - Consolidated Balance Sheets (Parenthetical)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_SeriesBConvertiblePreferredStockMember" xlink:label="loc_PULMSeriesBConvertiblePreferredStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PULMSeriesBConvertiblePreferredStockMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="loc_us-gaapPreferredStockParOrStatedValuePerShare_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockParOrStatedValuePerShare_50" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="loc_us-gaapPreferredStockSharesAuthorized_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockSharesAuthorized_50" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="loc_us-gaapPreferredStockSharesIssued_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockSharesIssued_50" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="loc_us-gaapPreferredStockSharesOutstanding_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockSharesOutstanding_50" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockConvertibleSharesIssuable" xlink:label="loc_us-gaapPreferredStockConvertibleSharesIssuable_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapPreferredStockConvertibleSharesIssuable_50" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="loc_us-gaapCommonStockParOrStatedValuePerShare_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockParOrStatedValuePerShare_50" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="loc_us-gaapCommonStockSharesAuthorized_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockSharesAuthorized_50" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesIssued" xlink:label="loc_us-gaapCommonStockSharesIssued_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockSharesIssued_50" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="loc_us-gaapCommonStockSharesOutstanding_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapCommonStockSharesOutstanding_50" xlink:type="arc" order="8" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/StatementsOfOperations" xlink:title="00000004 - Statement - Consolidated Statements of Operations (Unaudited)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/StatementsOfStockholdersEquity" xlink:title="00000005 - Statement - Consolidated Statements of Stockholders&apos; Equity (Unaudited)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems" xlink:label="loc_us-gaapStatementLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable" xlink:label="loc_us-gaapStatementTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStatementTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="loc_us-gaapStatementEquityComponentsAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementEquityComponentsAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain" xlink:label="loc_us-gaapEquityComponentDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementEquityComponentsAxis" xlink:to="loc_us-gaapEquityComponentDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockMember" xlink:label="loc_us-gaapPreferredStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapPreferredStockMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember" xlink:label="loc_us-gaapCommonStockMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapCommonStockMember_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="loc_us-gaapAdditionalPaidInCapitalMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapAdditionalPaidInCapitalMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember" xlink:label="loc_us-gaapRetainedEarningsMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapEquityComponentDomain" xlink:to="loc_us-gaapRetainedEarningsMember_30" xlink:type="arc" order="31" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapStatementTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_SeriesBConvertiblePreferredStockMember" xlink:label="loc_PULMSeriesBConvertiblePreferredStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PULMSeriesBConvertiblePreferredStockMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity" xlink:label="loc_us-gaapStockholdersEquity_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockholdersEquity_60" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesOutstanding" xlink:label="loc_us-gaapSharesOutstanding_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapSharesOutstanding_60" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodValueNewIssues_60" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="loc_us-gaapAdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapAdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_60" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss" xlink:label="loc_us-gaapNetIncomeLoss_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapStatementLineItems" xlink:to="loc_us-gaapNetIncomeLoss_60" xlink:type="arc" order="5" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/StatementsOfCashFlows" xlink:title="00000006 - Statement - Consolidated Statements of Cash Flows (Unaudited)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/Organization" xlink:title="999007 - Disclosure - Organization" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandards" xlink:title="999008 - Disclosure - Summary of Significant Accounting Policies and Recent Accounting Standards" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/PrepaidExpensesAndOtherCurrentAssets" xlink:title="999009 - Disclosure - Prepaid Expenses and Other Current Assets" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/AccruedExpensesAndOtherCurrentLiabilities" xlink:title="999010 - Disclosure - Accrued Expenses and Other Current Liabilities" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/PreferredStock" xlink:title="999011 - Disclosure - Preferred Stock" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/CommonStock" xlink:title="999012 - Disclosure - Common Stock" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/Warrants" xlink:title="999013 - Disclosure - Warrants" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/Stock-basedCompensation" xlink:title="999014 - Disclosure - Stock-based Compensation" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/CommitmentsAndContingencies" xlink:title="999015 - Disclosure - Commitments and Contingencies" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/IncomeTaxes" xlink:title="999016 - Disclosure - Income Taxes" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/NetLossPerShare" xlink:title="999017 - Disclosure - Net Loss Per Share" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/SegmentReporting" xlink:title="999018 - Disclosure - Segment Reporting" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/SubsequentEvents" xlink:title="999019 - Disclosure - Subsequent Events" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandardsPolicies" xlink:title="999020 - Disclosure - Summary of Significant Accounting Policies and Recent Accounting Standards (Policies)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/PrepaidExpensesAndOtherCurrentAssetsTables" xlink:title="999021 - Disclosure - Prepaid Expenses and Other Current Assets (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/AccruedExpensesAndOtherCurrentLiabilitiesTables" xlink:title="999022 - Disclosure - Accrued Expenses and Other Current Liabilities (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/WarrantsTables" xlink:title="999023 - Disclosure - Warrants (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/Stock-basedCompensationTables" xlink:title="999024 - Disclosure - Stock-based Compensation (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/NetLossPerShareTables" xlink:title="999025 - Disclosure - Net Loss Per Share (Tables)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/OrganizationDetailsNarrative" xlink:title="999026 - Disclosure - Organization (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:label="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:label="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_MergerAgreementMember" xlink:label="loc_PULMMergerAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_PULMMergerAgreementMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_OwnershipAxis" xlink:label="loc_srtOwnershipAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable" xlink:to="loc_srtOwnershipAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_OwnershipDomain" xlink:label="loc_srtOwnershipDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtOwnershipAxis" xlink:to="loc_srtOwnershipDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_PreMergerEosIncMember" xlink:label="loc_PULMPreMergerEosIncMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtOwnershipDomain" xlink:to="loc_PULMPreMergerEosIncMember_10" xlink:type="arc" order="12" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_PreMergerPulmatrixIncMember" xlink:label="loc_PULMPreMergerPulmatrixIncMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtOwnershipDomain" xlink:to="loc_PULMPreMergerPulmatrixIncMember_20" xlink:type="arc" order="22" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromOtherEquity" xlink:label="loc_us-gaapProceedsFromOtherEquity_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapProceedsFromOtherEquity_40" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromSaleOfOtherInvestments" xlink:label="loc_us-gaapProceedsFromSaleOfOtherInvestments_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapProceedsFromSaleOfOtherInvestments_40" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MinorityInterestOwnershipPercentageByNoncontrollingOwners" xlink:label="loc_us-gaapMinorityInterestOwnershipPercentageByNoncontrollingOwners_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapMinorityInterestOwnershipPercentageByNoncontrollingOwners_40" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_MinorityInterestOwnershipPercentageByParent" xlink:label="loc_us-gaapMinorityInterestOwnershipPercentageByParent_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems" xlink:to="loc_us-gaapMinorityInterestOwnershipPercentageByParent_40" xlink:type="arc" order="3" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/SummaryOfSignificantAccountingPoliciesAndRecentAccountingStandardsDetailsNarrative" xlink:title="999027 - Disclosure - Summary of Significant Accounting Policies and Recent Accounting Standards (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/ScheduleOfPrepaidExpensesAndOtherCurrentAssetsDetails" xlink:title="999028 - Disclosure - Schedule of Prepaid Expenses and Other Current Assets (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/ScheduleOfAccruedExpensesAndOtherCurrentLiabilitiesDetails" xlink:title="999029 - Disclosure - Schedule of Accrued Expenses and Other Current Liabilities (Details)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/PreferredStockDetailsNarrative" xlink:title="999030 - Disclosure - Preferred Stock (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockLineItems" xlink:label="loc_us-gaapClassOfStockLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:label="loc_us-gaapScheduleOfStockByClassTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapScheduleOfStockByClassTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="loc_us-gaapStatementClassOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfStockByClassTable" xlink:to="loc_us-gaapStatementClassOfStockAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfStockDomain" xlink:label="loc_us-gaapClassOfStockDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapStatementClassOfStockAxis" xlink:to="loc_us-gaapClassOfStockDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_SeriesAConvertiblePreferredStockMember" xlink:label="loc_PULMSeriesAConvertiblePreferredStockMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PULMSeriesAConvertiblePreferredStockMember_10" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_SeriesBConvertiblePreferredStockMember" xlink:label="loc_PULMSeriesBConvertiblePreferredStockMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockDomain" xlink:to="loc_PULMSeriesBConvertiblePreferredStockMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfStockByClassTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_MergerAgreementMember" xlink:label="loc_PULMMergerAgreementMember_50" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_PULMMergerAgreementMember_50" xlink:type="arc" order="52" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_PurchaseAgreementMember" xlink:label="loc_PULMPurchaseAgreementMember_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_PULMPurchaseAgreementMember_70" xlink:type="arc" order="71" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="loc_us-gaapPreferredStockSharesAuthorized_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapPreferredStockSharesAuthorized_100" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="loc_us-gaapPreferredStockParOrStatedValuePerShare_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapPreferredStockParOrStatedValuePerShare_100" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="loc_us-gaapPreferredStockSharesIssued_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapPreferredStockSharesIssued_100" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="loc_us-gaapPreferredStockSharesOutstanding_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapPreferredStockSharesOutstanding_100" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockValue" xlink:label="loc_us-gaapPreferredStockValue_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapPreferredStockValue_100" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapStockIssuedDuringPeriodSharesNewIssues_100" xlink:type="arc" order="5" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfConvertiblePreferredStock" xlink:label="loc_us-gaapProceedsFromIssuanceOfConvertiblePreferredStock_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapProceedsFromIssuanceOfConvertiblePreferredStock_100" xlink:type="arc" order="6" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockConvertibleConversionPrice" xlink:label="loc_us-gaapPreferredStockConvertibleConversionPrice_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapPreferredStockConvertibleConversionPrice_100" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PreferredStockDividendRatePercentage" xlink:label="loc_us-gaapPreferredStockDividendRatePercentage_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapPreferredStockDividendRatePercentage_100" xlink:type="arc" order="8" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DeferredFinanceCostsNet" xlink:label="loc_us-gaapDeferredFinanceCostsNet_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapDeferredFinanceCostsNet_100" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_ProceedsForWorkingCapital" xlink:label="loc_PULMProceedsForWorkingCapital_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_PULMProceedsForWorkingCapital_100" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromSaleOfRestrictedInvestments" xlink:label="loc_us-gaapProceedsFromSaleOfRestrictedInvestments_100" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapClassOfStockLineItems" xlink:to="loc_us-gaapProceedsFromSaleOfRestrictedInvestments_100" xlink:type="arc" order="11" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/CommonStockDetailsNarrative" xlink:title="999031 - Disclosure - Common Stock (Details Narrative)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockLineItems" xlink:label="loc_us-gaapSubsidiarySaleOfStockLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:label="loc_us-gaapSubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSubsidiarySaleOfStockLineItems" xlink:to="loc_us-gaapSubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_LegalEntityAxis" xlink:label="loc_deiLegalEntityAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:to="loc_deiLegalEntityAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2026/dei-2026.xsd#dei_EntityDomain" xlink:label="loc_deiEntityDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_deiLegalEntityAxis" xlink:to="loc_deiEntityDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_HCWainwrightAndCoLLCMember" xlink:label="loc_PULMHCWainwrightAndCoLLCMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_deiEntityDomain" xlink:to="loc_PULMHCWainwrightAndCoLLCMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="loc_us-gaapTypeOfArrangementAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:to="loc_us-gaapTypeOfArrangementAxis" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapTypeOfArrangementAxis" xlink:to="loc_us-gaapArrangementsAndNonarrangementTransactionsMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_SaleAgreementMember" xlink:label="loc_PULMSaleAgreementMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapArrangementsAndNonarrangementTransactionsMember" xlink:to="loc_PULMSaleAgreementMember_0" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis" xlink:label="loc_srtRangeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:to="loc_srtRangeAxis" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember" xlink:label="loc_srtRangeMember_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_srtRangeAxis" xlink:to="loc_srtRangeMember_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember" xlink:label="loc_srtMaximumMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srtRangeMember" xlink:to="loc_srtMaximumMember_0" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="loc_us-gaapSubsidiarySaleOfStockAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapSubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable" xlink:to="loc_us-gaapSubsidiarySaleOfStockAxis" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="loc_us-gaapSaleOfStockNameOfTransactionDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapSubsidiarySaleOfStockAxis" xlink:to="loc_us-gaapSaleOfStockNameOfTransactionDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="loc_us-gaapSaleOfStockNameOfTransactionDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapSubsidiarySaleOfStockAxis" xlink:to="loc_us-gaapSaleOfStockNameOfTransactionDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_ATMOfferingMember" xlink:label="loc_PULMATMOfferingMember_30" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSaleOfStockNameOfTransactionDomain" xlink:to="loc_PULMATMOfferingMember_30" xlink:type="arc" order="33" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction" xlink:label="loc_us-gaapSaleOfStockConsiderationReceivedOnTransaction_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsidiarySaleOfStockLineItems" xlink:to="loc_us-gaapSaleOfStockConsiderationReceivedOnTransaction_70" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockDescriptionOfTransaction" xlink:label="loc_us-gaapSaleOfStockDescriptionOfTransaction_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsidiarySaleOfStockLineItems" xlink:to="loc_us-gaapSaleOfStockDescriptionOfTransaction_70" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_EntitiesPublicFloat" xlink:label="loc_PULMEntitiesPublicFloat_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsidiarySaleOfStockLineItems" xlink:to="loc_PULMEntitiesPublicFloat_70" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_CommissionPercentage" xlink:label="loc_PULMCommissionPercentage_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsidiarySaleOfStockLineItems" xlink:to="loc_PULMCommissionPercentage_70" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapSubsidiarySaleOfStockLineItems" xlink:to="loc_us-gaapSaleOfStockNumberOfSharesIssuedInTransaction_70" xlink:type="arc" order="4" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/ScheduleOfWarrantsOutstandingAndExercisableDetails" xlink:title="999032 - Disclosure - Schedule of Warrants Outstanding and Exercisable (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis" xlink:label="loc_us-gaapAwardTypeAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="loc_us-gaapAwardTypeAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAwardTypeAxis" xlink:to="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_WarrantOneMember" xlink:label="loc_PULMWarrantOneMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_PULMWarrantOneMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_WarrantTwoMember" xlink:label="loc_PULMWarrantTwoMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_PULMWarrantTwoMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember" xlink:label="loc_us-gaapWarrantMember_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="loc_us-gaapWarrantMember_40" xlink:type="arc" order="41" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRighstDateFromWhichWarrantsOrRightsExercisable" xlink:label="loc_us-gaapClassOfWarrantOrRighstDateFromWhichWarrantsOrRightsExercisable_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRighstDateFromWhichWarrantsOrRightsExercisable_60" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_60" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstandingMaturityDate" xlink:label="loc_us-gaapWarrantsAndRightsOutstandingMaturityDate_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapWarrantsAndRightsOutstandingMaturityDate_60" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber_60" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsOutstandingNumber_60" xlink:type="arc" order="3" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/WarrantsDetailsNarrative" xlink:title="999033 - Disclosure - Warrants (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/ScheduleOfStockOptionActivityDetails" xlink:title="999034 - Disclosure - Schedule of Stock Option Activity (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OffsettingAssetsLineItems" xlink:label="loc_us-gaapOffsettingAssetsLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OffsettingAssetsTable" xlink:label="loc_us-gaapOffsettingAssetsTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapOffsettingAssetsTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeInstrumentRiskAxis" xlink:label="loc_us-gaapDerivativeInstrumentRiskAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapOffsettingAssetsTable" xlink:to="loc_us-gaapDerivativeInstrumentRiskAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="loc_us-gaapDerivativeContractTypeDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapDerivativeInstrumentRiskAxis" xlink:to="loc_us-gaapDerivativeContractTypeDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DerivativeContractTypeDomain" xlink:label="loc_us-gaapDerivativeContractTypeDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapDerivativeInstrumentRiskAxis" xlink:to="loc_us-gaapDerivativeContractTypeDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockOptionMember" xlink:label="loc_us-gaapStockOptionMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapDerivativeContractTypeDomain" xlink:to="loc_us-gaapStockOptionMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_40" xlink:type="arc" order="0" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_40" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_40" xlink:type="arc" order="2" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriod" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriod_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriod_40" xlink:type="arc" order="3" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriodWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriodWeightedAverageExercisePrice_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresAndExpirationsInPeriodWeightedAverageExercisePrice_40" xlink:type="arc" order="4" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_40" xlink:type="arc" order="7" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber_40" xlink:type="arc" order="9" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" xlink:label="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice_40" xlink:type="arc" order="10" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableWeightedAverageRemainingContractualTerm1_40" xlink:type="arc" order="11" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" xlink:label="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1_40" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapOffsettingAssetsLineItems" xlink:to="loc_us-gaapSharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1_40" xlink:type="arc" order="12" />
    </link:definitionLink>
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/Stock-basedCompensationDetailsNarrative" xlink:title="999035 - Disclosure - Stock-based Compensation (Details Narrative)" />
    <link:definitionLink xlink:type="extended" xlink:role="http://pulmatrix.com/role/ScheduleOfComputationOfAnti-dilutiveWeighted-averageSharesOutstandingDetails" xlink:title="999036 - Disclosure - Schedule of Computation of Anti-Dilutive Weighted-Average Shares Outstanding (Details)">
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:label="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="loc_us-gaapScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="loc_us-gaapScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="arc" order="10" xbrldt:closed="true" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaapScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="loc_us-gaapAntidilutiveSecuritiesNameDomain" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:type="arc" order="20" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="loc_us-gaapAntidilutiveSecuritiesNameDomain_10" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xbrldt:contextElement="segment" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="loc_us-gaapAntidilutiveSecuritiesNameDomain_10" xlink:type="arc" order="30" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_OptionsToPurchaseCommonStockMember" xlink:label="loc_PULMOptionsToPurchaseCommonStockMember_0" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:to="loc_PULMOptionsToPurchaseCommonStockMember_0" xlink:type="arc" order="1" />
      <link:loc xlink:type="locator" xlink:href="pulm-20260331.xsd#PULM_WarrantsToPurchaseCommonStockMember" xlink:label="loc_PULMWarrantsToPurchaseCommonStockMember_20" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesNameDomain" xlink:to="loc_PULMWarrantsToPurchaseCommonStockMember_20" xlink:type="arc" order="21" />
      <link:loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_70" />
      <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="loc_us-gaapAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_70" xlink:type="arc" order="0" />
    </link:definitionLink>
</link:linkbase>
