<?xml version="1.0" encoding="US-ASCII"?>
<!-- Generated by CompSci Transform (tm) - http://www.compsciresources.com -->
<!-- Created: Wed Mar 25 16:22:23 UTC 2026 -->
<linkbase xmlns="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd" xmlns:xbrldt="http://xbrl.org/2005/xbrldt">
  <roleRef xlink:type="simple" xlink:href="acnb-20251231.xsd#acnb_r_AdditionalDimensions" roleURI="http://www.acnb.com/role/AdditionalDimensions"/>
  <arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default"/>
  <arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain"/>
  <arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension"/>
  <arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member"/>
  <arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" arcroleURI="http://xbrl.org/int/dim/arcrole/all"/>
<!-- [WMV6][5MgjGSCGIbuCZrdsIuNKUR1OmPB/Sov6xAaXp8wHQQNeJGQvqpKvZflO/g7H5aKg5X7KqL4gIh6grVfQZ2ikURM5mx0gC/LZceT/OukWJF4Jvu4M/eqASMwgmj1gSUY8QTnYelN1dETdzAU8wU69HPcERhZukdiNSCZHkyUC/l2xnIe7SQeYz+ay0c7rNBu1d4Rx84LFRduquSgWFTg0vAvojdeLvKy5o58+4IWt3StVhDwQnvMUtp9KfV3Mxt8N7RBtqSWjjQUgCPniDwJfOFMC1v9eU7rGHWMLee30Q5lqX1Lj/NKTNvLRrA+F9XU=] CSR-->
  <definitionLink xlink:role="http://www.acnb.com/role/AdditionalDimensions" xlink:type="extended"/>
</linkbase>
