AbstractSince the introduction of white‐nose syndrome (WNS) in North America, numerous species of bat have dwindled in numbers. These declines observed are often species‐specific and thus provides opportunity for a natural experiment to test for shifts in diet through relaxed resource partitioning in bat communities post‐introduction of WNS. Acoustic monitoring at locations in Southern Ontario pre‐ (2009–2011) and post‐WNS (2012–2014) introduction showed an increase in activity of big brown bats (Eptesicus fuscus) corresponding to a decline in the activity of little brown bats (Myotis lucifugus). Next‐generation sequencing of bat stomachs and guano in Southern Ontario before and after WNS allowed for the characterization of diet changes of these species. Post‐WNS, E. fuscus consumed a wider breadth of prey and many of the insect species once consumed by M. lucifugus, including several pest insects. These results suggest that the introduction of WNS has resulted in relaxed interspecific competition within these bat communities in Southern Ontario.