<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2026 Workiva-->
<!--r:019efa38-be8f-7dd3-ab00-1418443716b4,g:75cc0028-267f-482c-a31f-8dea11247937-->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" 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:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFCHANGESINSTOCKHOLDERSDEFICITEQUITYunaudited" xlink:type="simple" xlink:href="oncy-20260630.xsd#CONDENSEDCONSOLIDATEDSTATEMENTSOFCHANGESINSTOCKHOLDERSDEFICITEQUITYunaudited"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFCHANGESINSTOCKHOLDERSDEFICITEQUITYunaudited" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_019efa38-bf5f-7cf8-982f-a8763fec0dba" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_019efa38-bf5f-74d6-900b-0ccde477f582" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_019efa38-bf5f-7cf8-982f-a8763fec0dba" xlink:to="loc_us-gaap_StatementTable_019efa38-bf5f-74d6-900b-0ccde477f582" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_019efa38-bf5f-7dbc-810a-320bec8f9dec" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_019efa38-bf5f-74d6-900b-0ccde477f582" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_019efa38-bf5f-7dbc-810a-320bec8f9dec" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-7dbc-810a-320bec8f9dec_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019efa38-bf5f-7dbc-810a-320bec8f9dec" xlink:to="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-7dbc-810a-320bec8f9dec_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-70be-8efe-08ca0854f9e3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_019efa38-bf5f-7dbc-810a-320bec8f9dec" xlink:to="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-70be-8efe-08ca0854f9e3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_019efa38-bf5f-7618-a874-4f3c96d29160" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-70be-8efe-08ca0854f9e3" xlink:to="loc_us-gaap_CommonStockMember_019efa38-bf5f-7618-a874-4f3c96d29160" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapitalMember_019efa38-bf5f-7018-8c2f-0c2b185d2776" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-70be-8efe-08ca0854f9e3" xlink:to="loc_us-gaap_AdditionalPaidInCapitalMember_019efa38-bf5f-7018-8c2f-0c2b185d2776" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019efa38-bf5f-70da-b9ba-cb6963e0d8aa" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-70be-8efe-08ca0854f9e3" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_019efa38-bf5f-70da-b9ba-cb6963e0d8aa" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_019efa38-bf5f-7c48-a2ea-0eeae5c843a7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_019efa38-bf5f-70be-8efe-08ca0854f9e3" xlink:to="loc_us-gaap_RetainedEarningsMember_019efa38-bf5f-7c48-a2ea-0eeae5c843a7" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019efa38-bf5f-7cf8-982f-a8763fec0dba" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_019efa38-bf5f-7017-a146-9e2c7f4c60b0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_019efa38-bf5f-7017-a146-9e2c7f4c60b0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_019efa38-bf5f-78ff-a096-9168547b5359" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_StockholdersEquity_019efa38-bf5f-78ff-a096-9168547b5359" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ComprehensiveIncomeNetOfTax_019efa38-bf5f-72f8-a0e5-48bc4a615459" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ComprehensiveIncomeNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_ComprehensiveIncomeNetOfTax_019efa38-bf5f-72f8-a0e5-48bc4a615459" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures_019efa38-bf5f-788a-8b79-0b1464e9183f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures_019efa38-bf5f-788a-8b79-0b1464e9183f" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_StockIssuedDuringPeriodSharesStandbyEquityPurchaseAgreementIssued_019fa41e-c6ef-7d6b-b08c-925782c2618a" xlink:href="oncy-20260630.xsd#oncy_StockIssuedDuringPeriodSharesStandbyEquityPurchaseAgreementIssued"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_oncy_StockIssuedDuringPeriodSharesStandbyEquityPurchaseAgreementIssued_019fa41e-c6ef-7d6b-b08c-925782c2618a" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_StockIssuedDuringPeriodValueStandbyEquityPurchaseAgreementIssued_019fa41e-c6ef-75cb-a544-fdec9d89de8b" xlink:href="oncy-20260630.xsd#oncy_StockIssuedDuringPeriodValueStandbyEquityPurchaseAgreementIssued"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_oncy_StockIssuedDuringPeriodValueStandbyEquityPurchaseAgreementIssued_019fa41e-c6ef-75cb-a544-fdec9d89de8b" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_019efa38-bf5f-79f6-b193-f8e6e15aa966" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_019efa38-bf5f-79f6-b193-f8e6e15aa966" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_019efa38-bf5f-74ab-9185-8f60ff10238b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_019efa38-bf5f-74ab-9185-8f60ff10238b" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesIssuedForServices_019efa38-bf5f-70e9-8e19-fec5b3ab26de" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesIssuedForServices"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesIssuedForServices_019efa38-bf5f-70e9-8e19-fec5b3ab26de" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueIssuedForServices_019efa38-bf5f-7e98-a524-0a3e7d39ec64" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueIssuedForServices"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueIssuedForServices_019efa38-bf5f-7e98-a524-0a3e7d39ec64" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalWarrantIssued_019efa38-bf5f-7b9a-bf59-91a91a302295" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalWarrantIssued"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalWarrantIssued_019efa38-bf5f-7b9a-bf59-91a91a302295" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_APICShareBasedPaymentArrangementReclassification_019efa38-bf5f-758f-99c2-ce0897ba5e4d" xlink:href="oncy-20260630.xsd#oncy_APICShareBasedPaymentArrangementReclassification"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_oncy_APICShareBasedPaymentArrangementReclassification_019efa38-bf5f-758f-99c2-ce0897ba5e4d" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_019efa38-bf5f-731b-ac04-1e3920b7540f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_019efa38-bf5f-7af8-a63f-3cb438d5ae03" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_019efa38-bf5f-731b-ac04-1e3920b7540f" xlink:type="arc" order="13"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_019efa38-bf5f-7286-be7f-ce820fc17c76" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_019efa38-bf5f-7f1e-87f8-973d554a5593" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFCASHFLOWSunaudited" xlink:type="simple" xlink:href="oncy-20260630.xsd#CONDENSEDCONSOLIDATEDSTATEMENTSOFCASHFLOWSunaudited"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFCASHFLOWSunaudited" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_019fa2eb-351c-7a5b-b7e0-a407c4573e69" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_StatementTable_019fa2eb-351c-7a5b-b7e0-a407c4573e69" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_019fa2eb-351c-70ba-885f-b642b9c6dafe" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_019fa2eb-351c-7a5b-b7e0-a407c4573e69" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_019fa2eb-351c-70ba-885f-b642b9c6dafe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019fa2eb-351c-70ba-885f-b642b9c6dafe_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_019fa2eb-351c-70ba-885f-b642b9c6dafe" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019fa2eb-351c-70ba-885f-b642b9c6dafe_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019fa2eb-351c-71c3-a284-9562a08f8871" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_019fa2eb-351c-70ba-885f-b642b9c6dafe" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019fa2eb-351c-71c3-a284-9562a08f8871" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_AtTheMarketOfferingAgreementMember_019fa2eb-351c-79a7-8c34-5908b4ab2f5f" xlink:href="oncy-20260630.xsd#oncy_AtTheMarketOfferingAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019fa2eb-351c-71c3-a284-9562a08f8871" xlink:to="loc_oncy_AtTheMarketOfferingAgreementMember_019fa2eb-351c-79a7-8c34-5908b4ab2f5f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_StandbyEquityPurchaseAgreementMember_019fa2eb-351c-71f8-a712-d5ac0f475ae2" xlink:href="oncy-20260630.xsd#oncy_StandbyEquityPurchaseAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019fa2eb-351c-71c3-a284-9562a08f8871" xlink:to="loc_oncy_StandbyEquityPurchaseAgreementMember_019fa2eb-351c-71f8-a712-d5ac0f475ae2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7cfe-b655-21c0becb1f50" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7cfe-b655-21c0becb1f50" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_019fa2eb-351c-7e78-be5b-3c90945cee24" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7cfe-b655-21c0becb1f50" xlink:to="loc_us-gaap_NetIncomeLoss_019fa2eb-351c-7e78-be5b-3c90945cee24" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7cfe-b655-21c0becb1f50" xlink:to="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_Depreciation_019fa2eb-351c-7c32-a0b2-de721e7826b9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_Depreciation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_us-gaap_Depreciation_019fa2eb-351c-7c32-a0b2-de721e7826b9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_NoncashLeaseExpense_019fa2eb-351c-7fe6-8748-51f633126686" xlink:href="oncy-20260630.xsd#oncy_NoncashLeaseExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_oncy_NoncashLeaseExpense_019fa2eb-351c-7fe6-8748-51f633126686" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensation_019fa2eb-351c-7ea9-9ec6-43c7dab0e557" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_us-gaap_ShareBasedCompensation_019fa2eb-351c-7ea9-9ec6-43c7dab0e557" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IssuanceOfStockAndWarrantsForServicesOrClaims_019fa2eb-351c-7ef4-b75e-7e86dfbb5d69" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IssuanceOfStockAndWarrantsForServicesOrClaims"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_us-gaap_IssuanceOfStockAndWarrantsForServicesOrClaims_019fa2eb-351c-7ef4-b75e-7e86dfbb5d69" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignCurrencyTransactionGainLossUnrealized_019fa2eb-351c-7ae4-bcf6-5c584be4eb81" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ForeignCurrencyTransactionGainLossUnrealized"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_us-gaap_ForeignCurrencyTransactionGainLossUnrealized_019fa2eb-351c-7ae4-bcf6-5c584be4eb81" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAdjustmentOfWarrants_019fa2eb-351c-7291-9f07-3541728f81c2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAdjustmentOfWarrants"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_us-gaap_FairValueAdjustmentOfWarrants_019fa2eb-351c-7291-9f07-3541728f81c2" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquitySecuritiesFvNiRealizedGainLoss_019fa2eb-351c-729e-bc1e-28a57a0f8074" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquitySecuritiesFvNiRealizedGainLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_us-gaap_EquitySecuritiesFvNiRealizedGainLoss_019fa2eb-351c-729e-bc1e-28a57a0f8074" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_IssuanceOfStockForCommitmentFees_019fa2eb-351c-77b3-add3-295f64d82568" xlink:href="oncy-20260630.xsd#oncy_IssuanceOfStockForCommitmentFees"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AdjustmentsNoncashItemsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7a34-b959-8c0796bc29a7" xlink:to="loc_oncy_IssuanceOfStockForCommitmentFees_019fa2eb-351c-77b3-add3-295f64d82568" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInOperatingCapitalAbstract_019fa2eb-351c-7237-a70e-712eabce659a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInOperatingCapitalAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7cfe-b655-21c0becb1f50" xlink:to="loc_us-gaap_IncreaseDecreaseInOperatingCapitalAbstract_019fa2eb-351c-7237-a70e-712eabce659a" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInOtherReceivables_019fa2eb-351c-767b-a9bc-287802a6dd2a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInOtherReceivables"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInOperatingCapitalAbstract_019fa2eb-351c-7237-a70e-712eabce659a" xlink:to="loc_us-gaap_IncreaseDecreaseInOtherReceivables_019fa2eb-351c-767b-a9bc-287802a6dd2a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInPrepaidExpense_019fa2eb-351c-714d-b592-c12e5a1ac4f5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInPrepaidExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInOperatingCapitalAbstract_019fa2eb-351c-7237-a70e-712eabce659a" xlink:to="loc_us-gaap_IncreaseDecreaseInPrepaidExpense_019fa2eb-351c-714d-b592-c12e5a1ac4f5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInAccountsPayable_019fa2eb-351c-7d89-96a4-17f7764b1354" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInAccountsPayable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInOperatingCapitalAbstract_019fa2eb-351c-7237-a70e-712eabce659a" xlink:to="loc_us-gaap_IncreaseDecreaseInAccountsPayable_019fa2eb-351c-7d89-96a4-17f7764b1354" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInAccruedLiabilities_019fa2eb-351c-704d-849b-018e676290e6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInAccruedLiabilities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInOperatingCapitalAbstract_019fa2eb-351c-7237-a70e-712eabce659a" xlink:to="loc_us-gaap_IncreaseDecreaseInAccruedLiabilities_019fa2eb-351c-704d-849b-018e676290e6" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInOperatingLeaseLiability_019fa2eb-351c-7a37-931b-3ce5c0d387f2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInOperatingLeaseLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInOperatingCapitalAbstract_019fa2eb-351c-7237-a70e-712eabce659a" xlink:to="loc_us-gaap_IncreaseDecreaseInOperatingLeaseLiability_019fa2eb-351c-7a37-931b-3ce5c0d387f2" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetCashProvidedByUsedInOperatingActivities_019fa2eb-351c-7564-86ad-3ef119191d7a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract_019fa2eb-351c-7cfe-b655-21c0becb1f50" xlink:to="loc_us-gaap_NetCashProvidedByUsedInOperatingActivities_019fa2eb-351c-7564-86ad-3ef119191d7a" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract_019fa2eb-351c-7ab3-923f-31a1b72818cd" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract_019fa2eb-351c-7ab3-923f-31a1b72818cd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsToAcquirePropertyPlantAndEquipment_019fa2eb-351c-7b47-be0f-37dd46e7f825" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsToAcquirePropertyPlantAndEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract_019fa2eb-351c-7ab3-923f-31a1b72818cd" xlink:to="loc_us-gaap_PaymentsToAcquirePropertyPlantAndEquipment_019fa2eb-351c-7b47-be0f-37dd46e7f825" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetCashProvidedByUsedInInvestingActivities_019fa2eb-351c-77a0-8045-7b44d5dbb177" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract_019fa2eb-351c-7ab3-923f-31a1b72818cd" xlink:to="loc_us-gaap_NetCashProvidedByUsedInInvestingActivities_019fa2eb-351c-77a0-8045-7b44d5dbb177" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract_019fa2eb-351c-7d5c-90bf-918d8a650e8f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract_019fa2eb-351c-7d5c-90bf-918d8a650e8f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_019fa2eb-351c-7aae-aae3-0558ddb1ab39" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract_019fa2eb-351c-7d5c-90bf-918d8a650e8f" xlink:to="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_019fa2eb-351c-7aae-aae3-0558ddb1ab39" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetCashProvidedByUsedInFinancingActivities_019fa2eb-351c-784e-9288-6734311a7dff" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract_019fa2eb-351c-7d5c-90bf-918d8a650e8f" xlink:to="loc_us-gaap_NetCashProvidedByUsedInFinancingActivities_019fa2eb-351c-784e-9288-6734311a7dff" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EffectOfExchangeRateOnCashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents_019fa2eb-351c-7b72-8627-9137e1e90b5a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EffectOfExchangeRateOnCashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_EffectOfExchangeRateOnCashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents_019fa2eb-351c-7b72-8627-9137e1e90b5a" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect_019fa2eb-351c-73ba-9b8f-b53cc01c6f85" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect_019fa2eb-351c-73ba-9b8f-b53cc01c6f85" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents_019fa2eb-351c-7efb-b966-c6a951ef3e77" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents_019fa2eb-351c-7efb-b966-c6a951ef3e77" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents_019fa2eb-351c-7498-8ea4-0ada4eb3e411" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalents"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SupplementalCashFlowInformationAbstract_019fca73-f7a0-7bc3-b72f-47df8d7c368a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SupplementalCashFlowInformationAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_019fa2eb-351c-7723-97dd-c49050d6d4c9" xlink:to="loc_us-gaap_SupplementalCashFlowInformationAbstract_019fca73-f7a0-7bc3-b72f-47df8d7c368a" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeTaxesPaidNet_019fca76-651f-7e50-ab33-67700b46b16b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeTaxesPaidNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SupplementalCashFlowInformationAbstract_019fca73-f7a0-7bc3-b72f-47df8d7c368a" xlink:to="loc_us-gaap_IncomeTaxesPaidNet_019fca76-651f-7e50-ab33-67700b46b16b" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/CapitalStockAttheMarketOfferingAgreementsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#CapitalStockAttheMarketOfferingAgreementsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/CapitalStockAttheMarketOfferingAgreementsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable_019efa38-bf5f-7b79-b830-4bec00222617" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable_019efa38-bf5f-7b79-b830-4bec00222617" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_019efa38-bf5f-7455-9882-dcfada84bd03" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable_019efa38-bf5f-7b79-b830-4bec00222617" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_019efa38-bf5f-7455-9882-dcfada84bd03" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019efa38-bf5f-7455-9882-dcfada84bd03_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_019efa38-bf5f-7455-9882-dcfada84bd03" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019efa38-bf5f-7455-9882-dcfada84bd03_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019efa38-bf5f-75bb-b5e3-94b46c29e2ca" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_019efa38-bf5f-7455-9882-dcfada84bd03" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019efa38-bf5f-75bb-b5e3-94b46c29e2ca" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_April2026AtTheMarketOfferingAgreementMember_019fa339-4f67-7847-8afa-ba52d62fea47" xlink:href="oncy-20260630.xsd#oncy_April2026AtTheMarketOfferingAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019efa38-bf5f-75bb-b5e3-94b46c29e2ca" xlink:to="loc_oncy_April2026AtTheMarketOfferingAgreementMember_019fa339-4f67-7847-8afa-ba52d62fea47" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_October2025AtTheMarketOfferingAgreementMember_019efa38-bf5f-7546-826a-4daa15bf0579" xlink:href="oncy-20260630.xsd#oncy_October2025AtTheMarketOfferingAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019efa38-bf5f-75bb-b5e3-94b46c29e2ca" xlink:to="loc_oncy_October2025AtTheMarketOfferingAgreementMember_019efa38-bf5f-7546-826a-4daa15bf0579" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_August2024AtTheMarketOfferingAgreementMember_019efa38-bf5f-76d0-abd7-fd55e56a2c53" xlink:href="oncy-20260630.xsd#oncy_August2024AtTheMarketOfferingAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_019efa38-bf5f-75bb-b5e3-94b46c29e2ca" xlink:to="loc_oncy_August2024AtTheMarketOfferingAgreementMember_019efa38-bf5f-76d0-abd7-fd55e56a2c53" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_019faaa5-ee95-76c5-a291-6c099850fb87" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsidiaryOrEquityMethodInvesteeSaleOfStockBySubsidiaryOrEquityInvesteeTable_019efa38-bf5f-7b79-b830-4bec00222617" xlink:to="loc_srt_CounterpartyNameAxis_019faaa5-ee95-76c5-a291-6c099850fb87" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_019faaa5-ee95-76c5-a291-6c099850fb87_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_019faaa5-ee95-76c5-a291-6c099850fb87" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_019faaa5-ee95-76c5-a291-6c099850fb87_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_019faaa5-ee95-757b-902d-0d1628fbc394" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_019faaa5-ee95-76c5-a291-6c099850fb87" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_019faaa5-ee95-757b-902d-0d1628fbc394" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_JefferiesLLCMember_019faaa6-1f5d-7902-88e1-d296814f6a70" xlink:href="oncy-20260630.xsd#oncy_JefferiesLLCMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_019faaa5-ee95-757b-902d-0d1628fbc394" xlink:to="loc_oncy_JefferiesLLCMember_019faaa6-1f5d-7902-88e1-d296814f6a70" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockParOrStatedValuePerShare_019fa39e-4c69-76d4-a3dd-cb21c2d522f1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockParOrStatedValuePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_us-gaap_CommonStockParOrStatedValuePerShare_019fa39e-4c69-76d4-a3dd-cb21c2d522f1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_SaleOfStockAuthorizedAmount_019fa39e-4c69-7386-b631-ee053c5e6216" xlink:href="oncy-20260630.xsd#oncy_SaleOfStockAuthorizedAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_oncy_SaleOfStockAuthorizedAmount_019fa39e-4c69-7386-b631-ee053c5e6216" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_SaleOfStockAuthorizedPeriod_019fa39e-4c69-715a-883b-cc1a342ec72f" xlink:href="oncy-20260630.xsd#oncy_SaleOfStockAuthorizedPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_oncy_SaleOfStockAuthorizedPeriod_019fa39e-4c69-715a-883b-cc1a342ec72f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_019fa39e-4c69-7005-ac0d-fa5e817d89b1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_019fa39e-4c69-7005-ac0d-fa5e817d89b1" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_019fa39e-4c69-7ea8-9b48-52001f8dabec" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_019fa39e-4c69-7ea8-9b48-52001f8dabec" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_SaleOfStockAveragePricePerShare_019fa39e-4c69-7949-8c84-7071154cf4f9" xlink:href="oncy-20260630.xsd#oncy_SaleOfStockAveragePricePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_oncy_SaleOfStockAveragePricePerShare_019fa39e-4c69-7949-8c84-7071154cf4f9" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_019fa39e-4c69-7ebf-b9b5-c326762b0b1b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_019fa39e-4c69-7ebf-b9b5-c326762b0b1b" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsOfStockIssuanceCosts_019fa39e-4c69-7ddb-9a00-1b9c8c39dce7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsOfStockIssuanceCosts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_us-gaap_PaymentsOfStockIssuanceCosts_019fa39e-4c69-7ddb-9a00-1b9c8c39dce7" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsForCommissions_019fa39e-4c69-78ec-a1e7-cef4fbb3d8ac" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsForCommissions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsidiarySaleOfStockLineItems_019efa38-bf5f-7731-8a52-37a33cfbe862" xlink:to="loc_us-gaap_PaymentsForCommissions_019fa39e-4c69-78ec-a1e7-cef4fbb3d8ac" xlink:type="arc" order="8"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/CapitalStockConsultantServicesSharesIssuedDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#CapitalStockConsultantServicesSharesIssuedDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/CapitalStockConsultantServicesSharesIssuedDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7ba2-af28-062622af852c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7f6c-9e0e-c0216c62a306" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7ba2-af28-062622af852c" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7f6c-9e0e-c0216c62a306" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-72c5-86f2-56b661e7f827" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7f6c-9e0e-c0216c62a306" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-72c5-86f2-56b661e7f827" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-72c5-86f2-56b661e7f827_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-72c5-86f2-56b661e7f827" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-72c5-86f2-56b661e7f827_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7cd8-a494-73d7e97d5d9c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-72c5-86f2-56b661e7f827" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7cd8-a494-73d7e97d5d9c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ConsultingServiceAgreementsMember_019efa38-bf5f-772e-861d-a4ef0160daae" xlink:href="oncy-20260630.xsd#oncy_ConsultingServiceAgreementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7cd8-a494-73d7e97d5d9c" xlink:to="loc_oncy_ConsultingServiceAgreementsMember_019efa38-bf5f-772e-861d-a4ef0160daae" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesIssuedForServices_019efa38-bf5f-77aa-9e91-4bc463017a62" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesIssuedForServices"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7ba2-af28-062622af852c" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesIssuedForServices_019efa38-bf5f-77aa-9e91-4bc463017a62" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueIssuedForServices_019efa38-bf5f-77d0-b1dc-95cd88bf8f96" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueIssuedForServices"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7ba2-af28-062622af852c" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueIssuedForServices_019efa38-bf5f-77d0-b1dc-95cd88bf8f96" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_SharesIssuedAveragePricePerShare_019efa38-bf5f-7732-a052-4ca7ce86ba7f" xlink:href="oncy-20260630.xsd#oncy_SharesIssuedAveragePricePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7ba2-af28-062622af852c" xlink:to="loc_oncy_SharesIssuedAveragePricePerShare_019efa38-bf5f-7732-a052-4ca7ce86ba7f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_019efa38-bf5f-74e1-a60f-8b6239cd8b81" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7ba2-af28-062622af852c" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_019efa38-bf5f-74e1-a60f-8b6239cd8b81" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PrepaidExpenseCurrent_019efa38-bf5f-7bc6-99ce-34dbd29b92b8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PrepaidExpenseCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7ba2-af28-062622af852c" xlink:to="loc_us-gaap_PrepaidExpenseCurrent_019efa38-bf5f-7bc6-99ce-34dbd29b92b8" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/CapitalStockCompensationWarrantsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#CapitalStockCompensationWarrantsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/CapitalStockCompensationWarrantsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7554-826a-a40dcb38370c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightTable_019efa38-bf5f-78d5-a515-9fe8b74708ad" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7554-826a-a40dcb38370c" xlink:to="loc_us-gaap_ClassOfWarrantOrRightTable_019efa38-bf5f-78d5-a515-9fe8b74708ad" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7b1b-8f8e-0d16b6a1ad21" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_019efa38-bf5f-78d5-a515-9fe8b74708ad" xlink:to="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7b1b-8f8e-0d16b6a1ad21" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-7b1b-8f8e-0d16b6a1ad21_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7b1b-8f8e-0d16b6a1ad21" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-7b1b-8f8e-0d16b6a1ad21_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-73ea-b3f7-dce6f79205cb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7b1b-8f8e-0d16b6a1ad21" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-73ea-b3f7-dce6f79205cb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_CompensationWarrantMember_019efa38-bf5f-74d7-8178-db9c367910ba" xlink:href="oncy-20260630.xsd#oncy_CompensationWarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-73ea-b3f7-dce6f79205cb" xlink:to="loc_oncy_CompensationWarrantMember_019efa38-bf5f-74d7-8178-db9c367910ba" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ClassOfWarrantOrRightIssued_019efa38-bf5f-7f6e-a35b-ee6d80ee4f95" xlink:href="oncy-20260630.xsd#oncy_ClassOfWarrantOrRightIssued"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7554-826a-a40dcb38370c" xlink:to="loc_oncy_ClassOfWarrantOrRightIssued_019efa38-bf5f-7f6e-a35b-ee6d80ee4f95" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight_019efa38-bf5f-7de7-9492-468ce0bff729" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7554-826a-a40dcb38370c" xlink:to="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight_019efa38-bf5f-7de7-9492-468ce0bff729" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_019efa38-bf5f-743a-a0bd-9677e3f42f8a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7554-826a-a40dcb38370c" xlink:to="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_019efa38-bf5f-743a-a0bd-9677e3f42f8a" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantsAndRightsOutstandingTerm_019efa38-bf5f-7c2d-85f3-f23909131df2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstandingTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7554-826a-a40dcb38370c" xlink:to="loc_us-gaap_WarrantsAndRightsOutstandingTerm_019efa38-bf5f-7c2d-85f3-f23909131df2" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightOutstanding_019efa38-bf5f-7b0c-b24a-bf127a548007" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7554-826a-a40dcb38370c" xlink:to="loc_us-gaap_ClassOfWarrantOrRightOutstanding_019efa38-bf5f-7b0c-b24a-bf127a548007" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/CapitalStockWarrantsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#CapitalStockWarrantsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/CapitalStockWarrantsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightTable_019efa38-bf5f-780a-8d4c-f5592d3bd65f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_us-gaap_ClassOfWarrantOrRightTable_019efa38-bf5f-780a-8d4c-f5592d3bd65f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7403-bc4c-fa5be960cabc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_019efa38-bf5f-780a-8d4c-f5592d3bd65f" xlink:to="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7403-bc4c-fa5be960cabc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-7403-bc4c-fa5be960cabc_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7403-bc4c-fa5be960cabc" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-7403-bc4c-fa5be960cabc_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-760f-aaad-82e487313925" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_019efa38-bf5f-7403-bc4c-fa5be960cabc" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-760f-aaad-82e487313925" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantMember_019efa38-bf5f-77e9-875f-e446191ee3d0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_019efa38-bf5f-760f-aaad-82e487313925" xlink:to="loc_us-gaap_WarrantMember_019efa38-bf5f-77e9-875f-e446191ee3d0" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight_019efa38-bf5f-70fb-a5e3-f368b18d409d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByEachWarrantOrRight_019efa38-bf5f-70fb-a5e3-f368b18d409d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_019efa38-bf5f-758c-88bd-b7ed99bee7e6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_019efa38-bf5f-758c-88bd-b7ed99bee7e6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantsAndRightsOutstandingTerm_019efa38-bf5f-7bb0-8deb-46c75921c546" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantsAndRightsOutstandingTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_us-gaap_WarrantsAndRightsOutstandingTerm_019efa38-bf5f-7bb0-8deb-46c75921c546" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_WarrantsAndRightsOutstandingThresholdConsecutiveTradingDaysWeightedAveragePrice_019efa38-bf5f-7f92-b4ad-5602a0185de4" xlink:href="oncy-20260630.xsd#oncy_WarrantsAndRightsOutstandingThresholdConsecutiveTradingDaysWeightedAveragePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_oncy_WarrantsAndRightsOutstandingThresholdConsecutiveTradingDaysWeightedAveragePrice_019efa38-bf5f-7f92-b4ad-5602a0185de4" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_WarrantsAndRightsOutstandingThresholdConsecutiveTradingDays_019efa38-bf5f-7e9c-9337-ce4672689b66" xlink:href="oncy-20260630.xsd#oncy_WarrantsAndRightsOutstandingThresholdConsecutiveTradingDays"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_oncy_WarrantsAndRightsOutstandingThresholdConsecutiveTradingDays_019efa38-bf5f-7e9c-9337-ce4672689b66" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_WarrantsAndRightsOutstandingThresholdTradingDays_019efa38-bf5f-791b-bea1-d9f83ad83a8b" xlink:href="oncy-20260630.xsd#oncy_WarrantsAndRightsOutstandingThresholdTradingDays"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_oncy_WarrantsAndRightsOutstandingThresholdTradingDays_019efa38-bf5f-791b-bea1-d9f83ad83a8b" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_WarrantsAndRightsOutstandingExpirationPeriod_019efa38-bf5f-7ad6-b3ff-2e15c8e0ab2a" xlink:href="oncy-20260630.xsd#oncy_WarrantsAndRightsOutstandingExpirationPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_oncy_WarrantsAndRightsOutstandingExpirationPeriod_019efa38-bf5f-7ad6-b3ff-2e15c8e0ab2a" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightOutstanding_019efa38-bf5f-7d9a-bfc6-ed8afbcd2809" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_019efa38-bf5f-7052-b6e1-df8f53cf36dc" xlink:to="loc_us-gaap_ClassOfWarrantOrRightOutstanding_019efa38-bf5f-7d9a-bfc6-ed8afbcd2809" xlink:type="arc" order="7"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/CapitalStockScheduleofCommonSharesReservedForFutureIssuanceDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#CapitalStockScheduleofCommonSharesReservedForFutureIssuanceDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/CapitalStockScheduleofCommonSharesReservedForFutureIssuanceDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019fa369-0a90-72fa-a0e4-030afd30f3dd" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019fa369-0a90-7bce-92a8-22126b2e6925" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019fa369-0a90-72fa-a0e4-030afd30f3dd" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019fa369-0a90-7bce-92a8-22126b2e6925" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019fa369-0a90-743f-84cf-e18e2d6c6fe1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019fa369-0a90-7bce-92a8-22126b2e6925" xlink:to="loc_us-gaap_AwardTypeAxis_019fa369-0a90-743f-84cf-e18e2d6c6fe1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-743f-84cf-e18e2d6c6fe1_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019fa369-0a90-743f-84cf-e18e2d6c6fe1" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-743f-84cf-e18e2d6c6fe1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-7ac4-87cf-ab78c577ba74" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019fa369-0a90-743f-84cf-e18e2d6c6fe1" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-7ac4-87cf-ab78c577ba74" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019fd7fe-f546-792a-bf11-032a5a63de0b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-7ac4-87cf-ab78c577ba74" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019fd7fe-f546-792a-bf11-032a5a63de0b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019fa369-0a90-72e8-bdbf-8826b31494af" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-7ac4-87cf-ab78c577ba74" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019fa369-0a90-72e8-bdbf-8826b31494af" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_InducementShareAwardsMember_019fa369-0a90-7f41-9472-edddec55edf2" xlink:href="oncy-20260630.xsd#oncy_InducementShareAwardsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-7ac4-87cf-ab78c577ba74" xlink:to="loc_oncy_InducementShareAwardsMember_019fa369-0a90-7f41-9472-edddec55edf2" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_A2026IncentiveAwardPlanMember_019fa449-9ef6-79f1-ba33-c7548259c5a5" xlink:href="oncy-20260630.xsd#oncy_A2026IncentiveAwardPlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-7ac4-87cf-ab78c577ba74" xlink:to="loc_oncy_A2026IncentiveAwardPlanMember_019fa449-9ef6-79f1-ba33-c7548259c5a5" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantMember_019fa369-0a90-7a31-bf67-25c83b234502" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019fa369-0a90-7ac4-87cf-ab78c577ba74" xlink:to="loc_us-gaap_WarrantMember_019fa369-0a90-7a31-bf67-25c83b234502" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_019fa369-0a90-7013-b415-f80f09702c8c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019fa369-0a90-72fa-a0e4-030afd30f3dd" xlink:to="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_019fa369-0a90-7013-b415-f80f09702c8c" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationNarrativeDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7a36-a915-e53f5792160c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7a36-a915-e53f5792160c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_019efa38-bf5f-73fc-bdc3-f459179402b6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7a36-a915-e53f5792160c" xlink:to="loc_us-gaap_PlanNameAxis_019efa38-bf5f-73fc-bdc3-f459179402b6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019efa38-bf5f-73fc-bdc3-f459179402b6_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_019efa38-bf5f-73fc-bdc3-f459179402b6" xlink:to="loc_us-gaap_PlanNameDomain_019efa38-bf5f-73fc-bdc3-f459179402b6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019efa38-bf5f-77a7-bc1d-23eaa76804cc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_019efa38-bf5f-73fc-bdc3-f459179402b6" xlink:to="loc_us-gaap_PlanNameDomain_019efa38-bf5f-77a7-bc1d-23eaa76804cc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_A2026IncentiveAwardPlanMember_019efa38-bf5f-70d6-ad4b-405ab5f3f5d8" xlink:href="oncy-20260630.xsd#oncy_A2026IncentiveAwardPlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_019efa38-bf5f-77a7-bc1d-23eaa76804cc" xlink:to="loc_oncy_A2026IncentiveAwardPlanMember_019efa38-bf5f-70d6-ad4b-405ab5f3f5d8" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f91-9cdf-ae890d484bdb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7a36-a915-e53f5792160c" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f91-9cdf-ae890d484bdb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7f91-9cdf-ae890d484bdb_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f91-9cdf-ae890d484bdb" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7f91-9cdf-ae890d484bdb_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7986-bfde-dc255fbff535" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f91-9cdf-ae890d484bdb" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7986-bfde-dc255fbff535" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember_019efa38-bf5f-734c-be62-8df8c8afcf73" xlink:href="oncy-20260630.xsd#oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7986-bfde-dc255fbff535" xlink:to="loc_oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember_019efa38-bf5f-734c-be62-8df8c8afcf73" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019efa38-bf5f-785f-a6a5-9d7f58fcd7f4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7986-bfde-dc255fbff535" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019efa38-bf5f-785f-a6a5-9d7f58fcd7f4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedPaymentArrangementOptionServiceConditionMember_019efa38-bf5f-7a66-b0e7-3e26ae95c3ed" xlink:href="oncy-20260630.xsd#oncy_ShareBasedPaymentArrangementOptionServiceConditionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7986-bfde-dc255fbff535" xlink:to="loc_oncy_ShareBasedPaymentArrangementOptionServiceConditionMember_019efa38-bf5f-7a66-b0e7-3e26ae95c3ed" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019efa38-bf5f-7f6a-a068-ed11ca5a1157" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7986-bfde-dc255fbff535" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019efa38-bf5f-7f6a-a068-ed11ca5a1157" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_InducementShareAwardsMember_019efa38-bf5f-7c91-9a1f-b51269fe7be9" xlink:href="oncy-20260630.xsd#oncy_InducementShareAwardsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7986-bfde-dc255fbff535" xlink:to="loc_oncy_InducementShareAwardsMember_019efa38-bf5f-7c91-9a1f-b51269fe7be9" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_019efa38-bf5f-797b-b3f1-cf82aa7c6ac1" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7a36-a915-e53f5792160c" xlink:to="loc_srt_RangeAxis_019efa38-bf5f-797b-b3f1-cf82aa7c6ac1" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_019efa38-bf5f-797b-b3f1-cf82aa7c6ac1_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_019efa38-bf5f-797b-b3f1-cf82aa7c6ac1" xlink:to="loc_srt_RangeMember_019efa38-bf5f-797b-b3f1-cf82aa7c6ac1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_019efa38-bf5f-7d32-83aa-8ad6c526374d" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_019efa38-bf5f-797b-b3f1-cf82aa7c6ac1" xlink:to="loc_srt_RangeMember_019efa38-bf5f-7d32-83aa-8ad6c526374d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_019efa38-bf5f-742f-b85d-e0cd79fcd855" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_019efa38-bf5f-7d32-83aa-8ad6c526374d" xlink:to="loc_srt_MinimumMember_019efa38-bf5f-742f-b85d-e0cd79fcd855" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_019efa38-bf5f-758e-ae17-7574f7d68ce1" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_019efa38-bf5f-7d32-83aa-8ad6c526374d" xlink:to="loc_srt_MaximumMember_019efa38-bf5f-758e-ae17-7574f7d68ce1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_TitleOfIndividualAxis_019efa38-bf5f-7c6f-a635-57733ee0c955" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_TitleOfIndividualAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7a36-a915-e53f5792160c" xlink:to="loc_srt_TitleOfIndividualAxis_019efa38-bf5f-7c6f-a635-57733ee0c955" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_019efa38-bf5f-7c6f-a635-57733ee0c955_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_TitleOfIndividualAxis_019efa38-bf5f-7c6f-a635-57733ee0c955" xlink:to="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_019efa38-bf5f-7c6f-a635-57733ee0c955_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_019efa38-bf5f-7827-903e-381b6dcb87dd" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_TitleOfIndividualAxis_019efa38-bf5f-7c6f-a635-57733ee0c955" xlink:to="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_019efa38-bf5f-7827-903e-381b6dcb87dd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ChiefExecutiveOfficerMember_019efa38-bf5f-7912-94bd-ce3285f8e5b3" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ChiefExecutiveOfficerMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_TitleOfIndividualWithRelationshipToEntityDomain_019efa38-bf5f-7827-903e-381b6dcb87dd" xlink:to="loc_srt_ChiefExecutiveOfficerMember_019efa38-bf5f-7912-94bd-ce3285f8e5b3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_019efa38-bf5f-70f8-a093-ea9248fb0f9b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_019efa38-bf5f-70f8-a093-ea9248fb0f9b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_CommonStockCapitalSharesReservedForFutureIssuanceAnnualIncreasePercentage_019efa38-bf5f-7489-adfe-2d2feaf4fa4d" xlink:href="oncy-20260630.xsd#oncy_CommonStockCapitalSharesReservedForFutureIssuanceAnnualIncreasePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_oncy_CommonStockCapitalSharesReservedForFutureIssuanceAnnualIncreasePercentage_019efa38-bf5f-7489-adfe-2d2feaf4fa4d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_019efa38-bf5f-71c4-a384-da6e93ceff4f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_019efa38-bf5f-71c4-a384-da6e93ceff4f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_019efa38-bf5f-784b-98f5-76b656f7df5b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_019efa38-bf5f-784b-98f5-76b656f7df5b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_019efa38-bf5f-7164-9c3a-c037efd98f64" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_019efa38-bf5f-7164-9c3a-c037efd98f64" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsThresholdForVestingMinimumAmount_019efa38-bf5f-73d5-b950-ef05d87816f6" xlink:href="oncy-20260630.xsd#oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsThresholdForVestingMinimumAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsThresholdForVestingMinimumAmount_019efa38-bf5f-73d5-b950-ef05d87816f6" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_019efa38-bf5f-7729-b88c-5582de0ba087" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_019efa38-bf5f-7729-b88c-5582de0ba087" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019efa38-bf5f-70a9-a2b8-abc8606cbd08" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019efa38-bf5f-70a9-a2b8-abc8606cbd08" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfOutstandingStockMaximum_019efa38-bf5f-7138-b7b3-d70b71881b55" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfOutstandingStockMaximum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardPercentageOfOutstandingStockMaximum_019efa38-bf5f-7138-b7b3-d70b71881b55" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumNumberOfSharesPerEmployee_019efa38-bf5f-7882-a6e0-4f977cbe2046" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumNumberOfSharesPerEmployee"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardMaximumNumberOfSharesPerEmployee_019efa38-bf5f-7882-a6e0-4f977cbe2046" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_019efa38-bf5f-7381-86cb-1f101e4f3003" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7f20-8a9d-f236f2a80121" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant_019efa38-bf5f-7381-86cb-1f101e4f3003" xlink:type="arc" order="10"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockBasedCompensationExpenseDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationScheduleofStockBasedCompensationExpenseDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockBasedCompensationExpenseDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-76bb-a69f-736d00966e7f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-76b1-a6ff-054354b490a8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-76bb-a69f-736d00966e7f" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-76b1-a6ff-054354b490a8" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-76b1-a6ff-054354b490a8" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-701b-b86e-4daba779e80d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-701b-b86e-4daba779e80d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockCompensationPlanMember_019efa38-bf5f-766c-9c41-8e40a57e2c4f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockCompensationPlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:to="loc_us-gaap_StockCompensationPlanMember_019efa38-bf5f-766c-9c41-8e40a57e2c4f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ConsultingServiceAgreementsMember_019efa38-bf5f-7892-bb32-8cc4f8c5445b" xlink:href="oncy-20260630.xsd#oncy_ConsultingServiceAgreementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:to="loc_oncy_ConsultingServiceAgreementsMember_019efa38-bf5f-7892-bb32-8cc4f8c5445b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementOfIncomeLocationBalanceAxis_019efa38-bf5f-7753-a201-95d5b2e6a06d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementOfIncomeLocationBalanceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-76b1-a6ff-054354b490a8" xlink:to="loc_us-gaap_StatementOfIncomeLocationBalanceAxis_019efa38-bf5f-7753-a201-95d5b2e6a06d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_019efa38-bf5f-73aa-822a-e68381966e3e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-76bb-a69f-736d00966e7f" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_019efa38-bf5f-73aa-822a-e68381966e3e" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockBasedCompensationExpenseDetails_1" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationScheduleofStockBasedCompensationExpenseDetails_1"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockBasedCompensationExpenseDetails_1" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-76bb-a69f-736d00966e7f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-76b1-a6ff-054354b490a8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-76bb-a69f-736d00966e7f" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-76b1-a6ff-054354b490a8" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-76b1-a6ff-054354b490a8" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-701b-b86e-4daba779e80d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-701b-b86e-4daba779e80d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-701b-b86e-4daba779e80d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockCompensationPlanMember_019efa38-bf5f-766c-9c41-8e40a57e2c4f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockCompensationPlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:to="loc_us-gaap_StockCompensationPlanMember_019efa38-bf5f-766c-9c41-8e40a57e2c4f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ConsultingServiceAgreementsMember_019efa38-bf5f-7892-bb32-8cc4f8c5445b" xlink:href="oncy-20260630.xsd#oncy_ConsultingServiceAgreementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-75d8-9664-8eb56899cef5" xlink:to="loc_oncy_ConsultingServiceAgreementsMember_019efa38-bf5f-7892-bb32-8cc4f8c5445b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_019efa38-bf5f-73aa-822a-e68381966e3e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-76bb-a69f-736d00966e7f" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_019efa38-bf5f-73aa-822a-e68381966e3e" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockOptionsSubjecttoServiceConditionsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationScheduleofStockOptionsSubjecttoServiceConditionsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockOptionsSubjecttoServiceConditionsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7a30-9d1b-f67488985f42" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7bde-a04d-77b8c593680d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7a30-9d1b-f67488985f42" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7bde-a04d-77b8c593680d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f6a-ae43-b93a5461afcf" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7bde-a04d-77b8c593680d" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f6a-ae43-b93a5461afcf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7f6a-ae43-b93a5461afcf_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f6a-ae43-b93a5461afcf" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7f6a-ae43-b93a5461afcf_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-722c-9a6e-e6d7754fcc91" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7f6a-ae43-b93a5461afcf" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-722c-9a6e-e6d7754fcc91" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedPaymentArrangementOptionServiceConditionMember_019efa38-bf5f-7b00-9e2d-30d8aad1ad16" xlink:href="oncy-20260630.xsd#oncy_ShareBasedPaymentArrangementOptionServiceConditionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-722c-9a6e-e6d7754fcc91" xlink:to="loc_oncy_ShareBasedPaymentArrangementOptionServiceConditionMember_019efa38-bf5f-7b00-9e2d-30d8aad1ad16" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember_019fb9b5-e69f-77c4-ad7e-0861cc26e7dd" xlink:href="oncy-20260630.xsd#oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-722c-9a6e-e6d7754fcc91" xlink:to="loc_oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember_019fb9b5-e69f-77c4-ad7e-0861cc26e7dd" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7c11-aa55-9728551b67d6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7bde-a04d-77b8c593680d" xlink:to="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7c11-aa55-9728551b67d6" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7c11-aa55-9728551b67d6_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7c11-aa55-9728551b67d6" xlink:to="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7c11-aa55-9728551b67d6_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7108-88b1-84ccc9bb9e65" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7c11-aa55-9728551b67d6" xlink:to="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7108-88b1-84ccc9bb9e65" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_InducementStockOptionsMember_019efa38-bf5f-737f-a3b6-4f0e4febb4b5" xlink:href="oncy-20260630.xsd#oncy_InducementStockOptionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7108-88b1-84ccc9bb9e65" xlink:to="loc_oncy_InducementStockOptionsMember_019efa38-bf5f-737f-a3b6-4f0e4febb4b5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-70b8-8872-17d691c05ab5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7a30-9d1b-f67488985f42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-70b8-8872-17d691c05ab5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf5f-7d49-8a2e-4c19b9b6d531" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-70b8-8872-17d691c05ab5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf5f-7d49-8a2e-4c19b9b6d531" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_019efa38-bf5f-7a93-a5c5-0b17df67a64f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-70b8-8872-17d691c05ab5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_019efa38-bf5f-7a93-a5c5-0b17df67a64f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod_019efa38-bf5f-7daa-af7f-b0ee8d3aec54" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-70b8-8872-17d691c05ab5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod_019efa38-bf5f-7daa-af7f-b0ee8d3aec54" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod_019efa38-bf5f-7cb7-b259-80c3ec6c65dc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-70b8-8872-17d691c05ab5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod_019efa38-bf5f-7cb7-b259-80c3ec6c65dc" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf5f-706f-a168-ba8eacb425e1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableNumber_019efa38-bf5f-78a3-b9f0-df77e95b6152" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7a30-9d1b-f67488985f42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableNumber_019efa38-bf5f-78a3-b9f0-df77e95b6152" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7f04-ab74-be689b4892e0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7a30-9d1b-f67488985f42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7f04-ab74-be689b4892e0" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_019efa38-bf5f-7b3a-a026-bdbb703488b4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7f04-ab74-be689b4892e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_019efa38-bf5f-7b3a-a026-bdbb703488b4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_019efa38-bf5f-7686-bfb2-35221ab8f5ca" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7f04-ab74-be689b4892e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_019efa38-bf5f-7686-bfb2-35221ab8f5ca" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice_019efa38-bf5f-76d7-b9de-56e62be56a56" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7f04-ab74-be689b4892e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice_019efa38-bf5f-76d7-b9de-56e62be56a56" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice_019efa38-bf5f-7013-afa7-333f50c25c0e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7f04-ab74-be689b4892e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice_019efa38-bf5f-7013-afa7-333f50c25c0e" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_019efa38-bf5f-745d-a825-355b3f1e2e66" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice_019efa38-bf5f-7225-8473-cf2d94bbb14b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7a30-9d1b-f67488985f42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice_019efa38-bf5f-7225-8473-cf2d94bbb14b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7098-9cb3-9a025dcdc306" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7a30-9d1b-f67488985f42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7098-9cb3-9a025dcdc306" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_019efa38-bf5f-7dc6-bb6a-dd9a4c471d7d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7098-9cb3-9a025dcdc306" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_019efa38-bf5f-7dc6-bb6a-dd9a4c471d7d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue_019efa38-bf5f-7ec8-b82f-a6c320775e1c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7098-9cb3-9a025dcdc306" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue_019efa38-bf5f-7ec8-b82f-a6c320775e1c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_019efa38-bf5f-72d1-a0fe-867c9d9368ba" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7098-9cb3-9a025dcdc306" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_019efa38-bf5f-72d1-a0fe-867c9d9368ba" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageRemainingContractualTerm1_019efa38-bf5f-70f1-b9d7-eafb200b0467" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageRemainingContractualTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7098-9cb3-9a025dcdc306" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageRemainingContractualTerm1_019efa38-bf5f-70f1-b9d7-eafb200b0467" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockOptionsSubjecttoPerformanceConditionsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationScheduleofStockOptionsSubjecttoPerformanceConditionsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockOptionsSubjecttoPerformanceConditionsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7eb7-92f6-55a5decb903d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7c66-9941-b288d0f4257e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7eb7-92f6-55a5decb903d" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7c66-9941-b288d0f4257e" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7c9d-9c3f-9f51aa7a42e0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7c66-9941-b288d0f4257e" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7c9d-9c3f-9f51aa7a42e0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7c9d-9c3f-9f51aa7a42e0_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7c9d-9c3f-9f51aa7a42e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7c9d-9c3f-9f51aa7a42e0_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7060-ac36-22d5c6bf720a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf5f-7c9d-9c3f-9f51aa7a42e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7060-ac36-22d5c6bf720a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember_019efa38-bf5f-7608-88da-e3a9c6966a00" xlink:href="oncy-20260630.xsd#oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf5f-7060-ac36-22d5c6bf720a" xlink:to="loc_oncy_ShareBasedPaymentArrangementOptionPerformanceConditionMember_019efa38-bf5f-7608-88da-e3a9c6966a00" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7e45-b9e0-fefb33b4accb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf5f-7c66-9941-b288d0f4257e" xlink:to="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7e45-b9e0-fefb33b4accb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7e45-b9e0-fefb33b4accb_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7e45-b9e0-fefb33b4accb" xlink:to="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7e45-b9e0-fefb33b4accb_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7ccf-a219-05d6fbf3252e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_019efa38-bf5f-7e45-b9e0-fefb33b4accb" xlink:to="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7ccf-a219-05d6fbf3252e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_InducementStockOptionsMember_019efa38-bf5f-7c5c-9c92-6c41fef5b30f" xlink:href="oncy-20260630.xsd#oncy_InducementStockOptionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_019efa38-bf5f-7ccf-a219-05d6fbf3252e" xlink:to="loc_oncy_InducementStockOptionsMember_019efa38-bf5f-7c5c-9c92-6c41fef5b30f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-7aa3-8267-7416d622ac4b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7eb7-92f6-55a5decb903d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-7aa3-8267-7416d622ac4b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf5f-7afc-be1b-60255134d70f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-7aa3-8267-7416d622ac4b" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf5f-7afc-be1b-60255134d70f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_019efa38-bf5f-7f85-b9b5-34fa9e4e2a5b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingRollForward_019efa38-bf5f-7aa3-8267-7416d622ac4b" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_019efa38-bf5f-7f85-b9b5-34fa9e4e2a5b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf5f-74f5-acb0-6c95710430db" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableNumber_019efa38-bf5f-78ac-a2e6-11057dc738d7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7eb7-92f6-55a5decb903d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableNumber_019efa38-bf5f-78ac-a2e6-11057dc738d7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7876-be81-e672a17819f3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7eb7-92f6-55a5decb903d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7876-be81-e672a17819f3" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_019efa38-bf5f-7597-8df0-b49af770b897" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7876-be81-e672a17819f3" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_019efa38-bf5f-7597-8df0-b49af770b897" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_019fa43d-2339-782b-9235-46e66da2292f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePriceRollforward_019efa38-bf5f-7876-be81-e672a17819f3" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice_019fa43d-2339-782b-9235-46e66da2292f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice_019efa38-bf5f-733f-8440-e13f80ed5ee9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice_019efa38-bf5f-7833-bb47-668a603ee90a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7eb7-92f6-55a5decb903d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageExercisePrice_019efa38-bf5f-7833-bb47-668a603ee90a" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7f3f-8248-4aabdd4c0c83" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf5f-7eb7-92f6-55a5decb903d" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7f3f-8248-4aabdd4c0c83" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_019efa38-bf5f-7f66-8724-19a9104dc310" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7f3f-8248-4aabdd4c0c83" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue_019efa38-bf5f-7f66-8724-19a9104dc310" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue_019efa38-bf5f-714e-96a5-adcd2f5bda23" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7f3f-8248-4aabdd4c0c83" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsVestedAndExpectedToVestExercisableAggregateIntrinsicValue_019efa38-bf5f-714e-96a5-adcd2f5bda23" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_019efa38-bf5f-7c08-9588-d21be7eaf3a2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7f3f-8248-4aabdd4c0c83" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsOutstandingWeightedAverageRemainingContractualTerm2_019efa38-bf5f-7c08-9588-d21be7eaf3a2" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageRemainingContractualTerm1_019efa38-bf5f-7c3e-b397-3a9b0eb8736e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageRemainingContractualTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsAdditionalDisclosuresAbstract_019efa38-bf5f-7f3f-8248-4aabdd4c0c83" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsVestedAndExpectedToVestExercisableWeightedAverageRemainingContractualTerm1_019efa38-bf5f-7c3e-b397-3a9b0eb8736e" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockOptionValuationAssumptionsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationScheduleofStockOptionValuationAssumptionsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofStockOptionValuationAssumptionsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-73ea-9104-ee2c31373f52" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-73ea-9104-ee2c31373f52" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7cf9-9ba9-5c3f4149f7c9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-73ea-9104-ee2c31373f52" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7cf9-9ba9-5c3f4149f7c9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7cf9-9ba9-5c3f4149f7c9_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7cf9-9ba9-5c3f4149f7c9" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7cf9-9ba9-5c3f4149f7c9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7e5c-9254-dd05af7e8fdc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7cf9-9ba9-5c3f4149f7c9" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7e5c-9254-dd05af7e8fdc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019efa38-bf60-7b1b-b72c-b44f757846e7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7e5c-9254-dd05af7e8fdc" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019efa38-bf60-7b1b-b72c-b44f757846e7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedPaymentArrangementLiabilityClassifiedOptionMember_019efa38-bf60-7b05-800f-1bfb729bcbc5" xlink:href="oncy-20260630.xsd#oncy_ShareBasedPaymentArrangementLiabilityClassifiedOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7e5c-9254-dd05af7e8fdc" xlink:to="loc_oncy_ShareBasedPaymentArrangementLiabilityClassifiedOptionMember_019efa38-bf60-7b05-800f-1bfb729bcbc5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_019efa38-bf60-71e0-80a4-a250e6373b51" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_019efa38-bf60-71e0-80a4-a250e6373b51" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_019efa38-bf60-7d1f-9412-c9c2c5e69c7c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_019efa38-bf60-7d1f-9412-c9c2c5e69c7c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_019efa38-bf60-78c4-96c8-b26c88b2605c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_019efa38-bf60-78c4-96c8-b26c88b2605c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageExpectedDividend_019efa38-bf60-7a0a-adc2-a6bd94435998" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageExpectedDividend"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageExpectedDividend_019efa38-bf60-7a0a-adc2-a6bd94435998" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice_019efa38-bf60-79e0-b177-a2ec2e70e36c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice_019efa38-bf60-79e0-b177-a2ec2e70e36c" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf60-708d-9bad-6dade693bec0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7768-bcbb-b0413863eb42" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber_019efa38-bf60-708d-9bad-6dade693bec0" xlink:type="arc" order="5"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleStockOptionLiabilityDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationScheduleStockOptionLiabilityDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleStockOptionLiabilityDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-751b-9539-4c20e1a863f7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-7a7f-b5c1-7aedd309586c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-751b-9539-4c20e1a863f7" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-7a7f-b5c1-7aedd309586c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7386-86c4-427323a482e0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-7a7f-b5c1-7aedd309586c" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7386-86c4-427323a482e0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7386-86c4-427323a482e0_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7386-86c4-427323a482e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7386-86c4-427323a482e0_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7fc7-ae2c-405473b8f5f3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7386-86c4-427323a482e0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7fc7-ae2c-405473b8f5f3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedPaymentArrangementLiabilityClassifiedOptionMember_019efa38-bf60-7d18-b8b6-abbdaac25e57" xlink:href="oncy-20260630.xsd#oncy_ShareBasedPaymentArrangementLiabilityClassifiedOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7fc7-ae2c-405473b8f5f3" xlink:to="loc_oncy_ShareBasedPaymentArrangementLiabilityClassifiedOptionMember_019efa38-bf60-7d18-b8b6-abbdaac25e57" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019fa3c1-0c09-757d-9481-be301e669fd3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7fc7-ae2c-405473b8f5f3" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019fa3c1-0c09-757d-9481-be301e669fd3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardStockOptionLiabilityRollForward_019efa38-bf60-77a3-8e84-cb769023658c" xlink:href="oncy-20260630.xsd#oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardStockOptionLiabilityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-751b-9539-4c20e1a863f7" xlink:to="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardStockOptionLiabilityRollForward_019efa38-bf60-77a3-8e84-cb769023658c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DeferredCompensationSharebasedArrangementsLiabilityClassifiedNoncurrent_019efa38-bf60-76c0-9408-8b67fd6f7a02" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DeferredCompensationSharebasedArrangementsLiabilityClassifiedNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardStockOptionLiabilityRollForward_019efa38-bf60-77a3-8e84-cb769023658c" xlink:to="loc_us-gaap_DeferredCompensationSharebasedArrangementsLiabilityClassifiedNoncurrent_019efa38-bf60-76c0-9408-8b67fd6f7a02" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_DeferredCompensationShareBasedArrangementsReclassificationOfLiabilityFromEquity_019efa38-bf60-77bb-89bf-1e449a90b7ea" xlink:href="oncy-20260630.xsd#oncy_DeferredCompensationShareBasedArrangementsReclassificationOfLiabilityFromEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardStockOptionLiabilityRollForward_019efa38-bf60-77a3-8e84-cb769023658c" xlink:to="loc_oncy_DeferredCompensationShareBasedArrangementsReclassificationOfLiabilityFromEquity_019efa38-bf60-77bb-89bf-1e449a90b7ea" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_DeferredCompensationShareBasedArrangementsShareBasedPaymentArrangementExpense_019efa38-bf60-7153-98c8-b440e48ff555" xlink:href="oncy-20260630.xsd#oncy_DeferredCompensationShareBasedArrangementsShareBasedPaymentArrangementExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardStockOptionLiabilityRollForward_019efa38-bf60-77a3-8e84-cb769023658c" xlink:to="loc_oncy_DeferredCompensationShareBasedArrangementsShareBasedPaymentArrangementExpense_019efa38-bf60-7153-98c8-b440e48ff555" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_DeferredCompensationShareBasedArrangementsAdjustmentToLiability_019efa38-bf60-789f-a35f-42bd3d6f227e" xlink:href="oncy-20260630.xsd#oncy_DeferredCompensationShareBasedArrangementsAdjustmentToLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_oncy_ShareBasedCompensationArrangementByShareBasedPaymentAwardStockOptionLiabilityRollForward_019efa38-bf60-77a3-8e84-cb769023658c" xlink:to="loc_oncy_DeferredCompensationShareBasedArrangementsAdjustmentToLiability_019efa38-bf60-789f-a35f-42bd3d6f227e" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DeferredCompensationSharebasedArrangementsLiabilityClassifiedNoncurrent_019efa38-bf60-74e4-9a5d-5cb106ab23cc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DeferredCompensationSharebasedArrangementsLiabilityClassifiedNoncurrent"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofRestrictedStockUnitsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#StockBasedCompensationScheduleofRestrictedStockUnitsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/StockBasedCompensationScheduleofRestrictedStockUnitsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7ef2-bfb6-3f1e48dbfd8e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-7ee0-a37f-1816fd8bb910" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7ef2-bfb6-3f1e48dbfd8e" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-7ee0-a37f-1816fd8bb910" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7ae2-a7e9-11149bffddca" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_019efa38-bf60-7ee0-a37f-1816fd8bb910" xlink:to="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7ae2-a7e9-11149bffddca" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7ae2-a7e9-11149bffddca_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7ae2-a7e9-11149bffddca" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7ae2-a7e9-11149bffddca_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7597-bc4e-59f2fd444b2b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_019efa38-bf60-7ae2-a7e9-11149bffddca" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7597-bc4e-59f2fd444b2b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_019efa38-bf60-7c88-8e4b-0c80961c1b16" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_019efa38-bf60-7597-bc4e-59f2fd444b2b" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_019efa38-bf60-7c88-8e4b-0c80961c1b16" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019efa38-bf60-7ac2-b178-5d1f9b6c47d5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7ef2-bfb6-3f1e48dbfd8e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019efa38-bf60-7ac2-b178-5d1f9b6c47d5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_019efa38-bf60-7abc-89df-a0e9f9cef227" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019efa38-bf60-7ac2-b178-5d1f9b6c47d5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_019efa38-bf60-7abc-89df-a0e9f9cef227" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_019efa38-bf60-7568-ba84-b481b598ea76" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019efa38-bf60-7ac2-b178-5d1f9b6c47d5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_019efa38-bf60-7568-ba84-b481b598ea76" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019efa38-bf60-74ee-9ff9-c1786abb1f5f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019efa38-bf60-7ac2-b178-5d1f9b6c47d5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_019efa38-bf60-74ee-9ff9-c1786abb1f5f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_019fa3c9-bebf-70a6-b459-de8a28974c0a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_019efa38-bf60-7ac2-b178-5d1f9b6c47d5" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_019fa3c9-bebf-70a6-b459-de8a28974c0a" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_019efa38-bf60-76ed-99ca-befeaf769920" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019efa38-bf60-70e1-99d1-b80e56e67164" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7ef2-bfb6-3f1e48dbfd8e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019efa38-bf60-70e1-99d1-b80e56e67164" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_019efa38-bf60-7e3f-add1-1dd4135fa1ef" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019efa38-bf60-70e1-99d1-b80e56e67164" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_019efa38-bf60-7e3f-add1-1dd4135fa1ef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_019efa38-bf60-7835-a597-f5edc99073f1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019efa38-bf60-70e1-99d1-b80e56e67164" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_019efa38-bf60-7835-a597-f5edc99073f1" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_019efa38-bf60-7c8c-bc6f-0dcd87523fc6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019efa38-bf60-70e1-99d1-b80e56e67164" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_019efa38-bf60-7c8c-bc6f-0dcd87523fc6" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_019fa3cb-e8a9-7d11-9e56-5940f6be3c03" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_019efa38-bf60-70e1-99d1-b80e56e67164" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_019fa3cb-e8a9-7d11-9e56-5940f6be3c03" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_019efa38-bf60-7813-9357-0ad73ecf4b52" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueAbstract_019efa38-bf60-7e6f-a90e-4b64bbb7e8ff" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_019efa38-bf60-7ef2-bfb6-3f1e48dbfd8e" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueAbstract_019efa38-bf60-7e6f-a90e-4b64bbb7e8ff" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueNonvested_019efa38-bf60-7ef0-b310-8266d4ee6e44" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueNonvested"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueAbstract_019efa38-bf60-7e6f-a90e-4b64bbb7e8ff" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueNonvested_019efa38-bf60-7ef0-b310-8266d4ee6e44" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueVested_019efa38-bf60-7f3b-a764-c149d02ce52e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueVested"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueAbstract_019efa38-bf60-7e6f-a90e-4b64bbb7e8ff" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardEquityInstrumentsOtherThanOptionsAggregateIntrinsicValueVested_019efa38-bf60-7f3b-a764-c149d02ce52e" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/NetLossPerShareScheduleofAntidilutiveSecuritiesExcludedfromComputationofNetLossPerShareDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#NetLossPerShareScheduleofAntidilutiveSecuritiesExcludedfromComputationofNetLossPerShareDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/NetLossPerShareScheduleofAntidilutiveSecuritiesExcludedfromComputationofNetLossPerShareDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019efa38-bf60-7a7f-8ea7-0054aa872b85" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019efa38-bf60-72f5-b365-52d98c2071a0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019efa38-bf60-7a7f-8ea7-0054aa872b85" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019efa38-bf60-72f5-b365-52d98c2071a0" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019efa38-bf60-7a00-9c6e-3b06c4e6fb1e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_019efa38-bf60-72f5-b365-52d98c2071a0" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019efa38-bf60-7a00-9c6e-3b06c4e6fb1e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019efa38-bf60-7a00-9c6e-3b06c4e6fb1e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019efa38-bf60-7a00-9c6e-3b06c4e6fb1e" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019efa38-bf60-7a00-9c6e-3b06c4e6fb1e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019efa38-bf60-7ec0-ba9a-a8350206c37c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_019efa38-bf60-7a00-9c6e-3b06c4e6fb1e" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019efa38-bf60-7ec0-ba9a-a8350206c37c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantMember_019efa38-bf60-767e-90b5-0d4e9a8bcdca" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019efa38-bf60-7ec0-ba9a-a8350206c37c" xlink:to="loc_us-gaap_WarrantMember_019efa38-bf60-767e-90b5-0d4e9a8bcdca" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_019efa38-bf60-7cdc-b4a8-4ea9659fc3ca" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019efa38-bf60-7ec0-ba9a-a8350206c37c" xlink:to="loc_us-gaap_EmployeeStockOptionMember_019efa38-bf60-7cdc-b4a8-4ea9659fc3ca" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_RestrictedStockUnitsAndIncentiveShareAwardsMember_019efa38-bf60-736c-9226-1a120a1f0d2b" xlink:href="oncy-20260630.xsd#oncy_RestrictedStockUnitsAndIncentiveShareAwardsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_019efa38-bf60-7ec0-ba9a-a8350206c37c" xlink:to="loc_oncy_RestrictedStockUnitsAndIncentiveShareAwardsMember_019efa38-bf60-736c-9226-1a120a1f0d2b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_019efa38-bf60-7b44-a95b-8d25ddb95fd0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_019efa38-bf60-7a7f-8ea7-0054aa872b85" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_019efa38-bf60-7b44-a95b-8d25ddb95fd0" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/ContractLiabilityDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#ContractLiabilityDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/ContractLiabilityDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019efa38-bf60-7130-bf06-71e414c5069b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019efa38-bf60-7629-9f6c-6a7dc284e260" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019efa38-bf60-7130-bf06-71e414c5069b" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019efa38-bf60-7629-9f6c-6a7dc284e260" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionStartDateAxis_019efa38-bf60-7104-a000-74521439839a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionStartDateAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019efa38-bf60-7629-9f6c-6a7dc284e260" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionStartDateAxis_019efa38-bf60-7104-a000-74521439839a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019efa38-bf60-717c-b196-e4639e2f81fb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019efa38-bf60-7130-bf06-71e414c5069b" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019efa38-bf60-717c-b196-e4639e2f81fb" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/ContractLiabilityDetails_1" xlink:type="simple" xlink:href="oncy-20260630.xsd#ContractLiabilityDetails_1"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/ContractLiabilityDetails_1" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019efa38-bf60-7130-bf06-71e414c5069b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019efa38-bf60-7629-9f6c-6a7dc284e260" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019efa38-bf60-7130-bf06-71e414c5069b" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionTable_019efa38-bf60-7629-9f6c-6a7dc284e260" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019efa38-bf60-717c-b196-e4639e2f81fb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionLineItems_019efa38-bf60-7130-bf06-71e414c5069b" xlink:to="loc_us-gaap_RevenueRemainingPerformanceObligationExpectedTimingOfSatisfactionPeriod1_019efa38-bf60-717c-b196-e4639e2f81fb" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/SegmentInformationScheduleofSegmentInformationDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#SegmentInformationScheduleofSegmentInformationDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/SegmentInformationScheduleofSegmentInformationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingInformationLineItems_019efa38-bf60-779d-9cf1-f9fa99c76270" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentReportingInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019efa38-bf60-7acc-8351-31e0cf3c74b5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019efa38-bf60-779d-9cf1-f9fa99c76270" xlink:to="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019efa38-bf60-7acc-8351-31e0cf3c74b5" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementBusinessSegmentsAxis_019efa38-bf60-7c94-afd3-f9752c0ce900" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementBusinessSegmentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_019efa38-bf60-7acc-8351-31e0cf3c74b5" xlink:to="loc_us-gaap_StatementBusinessSegmentsAxis_019efa38-bf60-7c94-afd3-f9752c0ce900" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019efa38-bf60-7c94-afd3-f9752c0ce900_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019efa38-bf60-7c94-afd3-f9752c0ce900" xlink:to="loc_us-gaap_SegmentDomain_019efa38-bf60-7c94-afd3-f9752c0ce900_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_019efa38-bf60-7fd6-abc0-364a04089fe7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_019efa38-bf60-7c94-afd3-f9752c0ce900" xlink:to="loc_us-gaap_SegmentDomain_019efa38-bf60-7fd6-abc0-364a04089fe7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ReportableSegmentMember_019efa38-bf60-7bdf-95cb-8f7340ebca71" xlink:href="oncy-20260630.xsd#oncy_ReportableSegmentMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_019efa38-bf60-7fd6-abc0-364a04089fe7" xlink:to="loc_oncy_ReportableSegmentMember_019efa38-bf60-7bdf-95cb-8f7340ebca71" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpensesAbstract_019efa38-bf60-7c18-b195-88e4a48991c3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingExpensesAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019efa38-bf60-779d-9cf1-f9fa99c76270" xlink:to="loc_us-gaap_OperatingExpensesAbstract_019efa38-bf60-7c18-b195-88e4a48991c3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_019efa38-bf60-7ffe-885c-1316cb4cdc82" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ResearchAndDevelopmentExpenseAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_019efa38-bf60-7c18-b195-88e4a48991c3" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_019efa38-bf60-7ffe-885c-1316cb4cdc82" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ResearchAndDevelopmentExpenseClinicalTrial_019efa38-bf60-72eb-ba93-c922d46b3021" xlink:href="oncy-20260630.xsd#oncy_ResearchAndDevelopmentExpenseClinicalTrial"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_019efa38-bf60-7ffe-885c-1316cb4cdc82" xlink:to="loc_oncy_ResearchAndDevelopmentExpenseClinicalTrial_019efa38-bf60-72eb-ba93-c922d46b3021" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_ResearchAndDevelopmentExpenseManufacturingAndRelatedProcessDevelopment_019efa38-bf60-7bfc-a869-fbb7485104ab" xlink:href="oncy-20260630.xsd#oncy_ResearchAndDevelopmentExpenseManufacturingAndRelatedProcessDevelopment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_019efa38-bf60-7ffe-885c-1316cb4cdc82" xlink:to="loc_oncy_ResearchAndDevelopmentExpenseManufacturingAndRelatedProcessDevelopment_019efa38-bf60-7bfc-a869-fbb7485104ab" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LaborAndRelatedExpense_019efa38-bf60-7445-832a-2f3e2303e6b5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LaborAndRelatedExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_019efa38-bf60-7ffe-885c-1316cb4cdc82" xlink:to="loc_us-gaap_LaborAndRelatedExpense_019efa38-bf60-7445-832a-2f3e2303e6b5" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GeneralAndAdministrativeExpenseAbstract_019efa38-bf60-71f3-8888-0955d53c883d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GeneralAndAdministrativeExpenseAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_019efa38-bf60-7c18-b195-88e4a48991c3" xlink:to="loc_us-gaap_GeneralAndAdministrativeExpenseAbstract_019efa38-bf60-71f3-8888-0955d53c883d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProfessionalFees_019efa38-bf60-7e4c-99df-9407e5ee17c7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProfessionalFees"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_GeneralAndAdministrativeExpenseAbstract_019efa38-bf60-71f3-8888-0955d53c883d" xlink:to="loc_us-gaap_ProfessionalFees_019efa38-bf60-7e4c-99df-9407e5ee17c7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SalariesWagesAndOfficersCompensation_019efa38-bf60-7817-b159-bb769dc9d28c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SalariesWagesAndOfficersCompensation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_GeneralAndAdministrativeExpenseAbstract_019efa38-bf60-71f3-8888-0955d53c883d" xlink:to="loc_us-gaap_SalariesWagesAndOfficersCompensation_019efa38-bf60-7817-b159-bb769dc9d28c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_IntellectualPropertyExpense_019efa38-bf60-7a75-b14d-b1808b9b459b" xlink:href="oncy-20260630.xsd#oncy_IntellectualPropertyExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_GeneralAndAdministrativeExpenseAbstract_019efa38-bf60-71f3-8888-0955d53c883d" xlink:to="loc_oncy_IntellectualPropertyExpense_019efa38-bf60-7a75-b14d-b1808b9b459b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_oncy_OtherResearchAndDevelopmentExpenseAndOtherGeneralAndAdministrativeExpense_019efa38-bf60-7a6d-b032-a2c844ae2644" xlink:href="oncy-20260630.xsd#oncy_OtherResearchAndDevelopmentExpenseAndOtherGeneralAndAdministrativeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_GeneralAndAdministrativeExpenseAbstract_019efa38-bf60-71f3-8888-0955d53c883d" xlink:to="loc_oncy_OtherResearchAndDevelopmentExpenseAndOtherGeneralAndAdministrativeExpense_019efa38-bf60-7a6d-b032-a2c844ae2644" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingExpenses_019efa38-bf60-7c6f-a168-57a7cc867524" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_OperatingExpensesAbstract_019efa38-bf60-7c18-b195-88e4a48991c3" xlink:to="loc_us-gaap_OperatingExpenses_019efa38-bf60-7c6f-a168-57a7cc867524" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingIncomeLoss_019efa38-bf60-7d8f-b513-c9194315de62" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019efa38-bf60-779d-9cf1-f9fa99c76270" xlink:to="loc_us-gaap_OperatingIncomeLoss_019efa38-bf60-7d8f-b513-c9194315de62" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherNonoperatingIncomeExpense_019efa38-bf60-7c76-abb1-4e464c694381" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherNonoperatingIncomeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019efa38-bf60-779d-9cf1-f9fa99c76270" xlink:to="loc_us-gaap_OtherNonoperatingIncomeExpense_019efa38-bf60-7c76-abb1-4e464c694381" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeTaxExpenseBenefit_019fa352-0285-79f3-8fd6-129d0b596a90" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncomeTaxExpenseBenefit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019efa38-bf60-779d-9cf1-f9fa99c76270" xlink:to="loc_us-gaap_IncomeTaxExpenseBenefit_019fa352-0285-79f3-8fd6-129d0b596a90" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_019efa38-bf60-7556-a637-38c4ed5a1f42" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_019efa38-bf60-779d-9cf1-f9fa99c76270" xlink:to="loc_us-gaap_NetIncomeLoss_019efa38-bf60-7556-a637-38c4ed5a1f42" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.oncolyticsbiotech.com/role/SubsequentEventsDetails" xlink:type="simple" xlink:href="oncy-20260630.xsd#SubsequentEventsDetails"/>
  <link:definitionLink xlink:role="http://www.oncolyticsbiotech.com/role/SubsequentEventsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventLineItems_019efa38-bf60-723a-8e4e-c31d24cb607b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTable_019efa38-bf60-7390-97c5-145404f992f1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SubsequentEventLineItems_019efa38-bf60-723a-8e4e-c31d24cb607b" xlink:to="loc_us-gaap_SubsequentEventTable_019efa38-bf60-7390-97c5-145404f992f1" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeAxis_019efa38-bf60-7b5e-b8d5-59764a594de9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_019efa38-bf60-7390-97c5-145404f992f1" xlink:to="loc_us-gaap_SubsequentEventTypeAxis_019efa38-bf60-7b5e-b8d5-59764a594de9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_019efa38-bf60-7b5e-b8d5-59764a594de9_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_019efa38-bf60-7b5e-b8d5-59764a594de9" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_019efa38-bf60-7b5e-b8d5-59764a594de9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_019efa38-bf60-71b2-970b-d6198ef7dada" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_019efa38-bf60-7b5e-b8d5-59764a594de9" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_019efa38-bf60-71b2-970b-d6198ef7dada" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventMember_019efa38-bf60-787a-8a29-2a4b8604f8eb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventTypeDomain_019efa38-bf60-71b2-970b-d6198ef7dada" xlink:to="loc_us-gaap_SubsequentEventMember_019efa38-bf60-787a-8a29-2a4b8604f8eb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_019efa38-bf60-7d06-a8c3-6410a37108a5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_019efa38-bf60-723a-8e4e-c31d24cb607b" xlink:to="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_019efa38-bf60-7d06-a8c3-6410a37108a5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_019efa38-bf60-7173-a61e-a721e2877e2b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_019efa38-bf60-723a-8e4e-c31d24cb607b" xlink:to="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_019efa38-bf60-7173-a61e-a721e2877e2b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockPricePerShare_019efa38-bf60-77ee-b3cc-23e484d68e4b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockPricePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_019efa38-bf60-723a-8e4e-c31d24cb607b" xlink:to="loc_us-gaap_SaleOfStockPricePerShare_019efa38-bf60-77ee-b3cc-23e484d68e4b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_019efa38-bf60-7a1c-80ac-892618ae1f5c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_019efa38-bf60-723a-8e4e-c31d24cb607b" xlink:to="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_019efa38-bf60-7a1c-80ac-892618ae1f5c" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsOfStockIssuanceCosts_019efa38-bf60-762b-af98-d1830dfe637d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PaymentsOfStockIssuanceCosts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_019efa38-bf60-723a-8e4e-c31d24cb607b" xlink:to="loc_us-gaap_PaymentsOfStockIssuanceCosts_019efa38-bf60-762b-af98-d1830dfe637d" xlink:type="arc" order="4"/>
  </link:definitionLink>
</link:linkbase>
