High-throughput DNA sequencing can generate large genetic datasets in a cost-effective manner. Although the diet of Eptesicus fuscus (Big Brown Bat) has been studied widely in natural and rural systems using visual identification of prey items in feces, our aim was to more completely assess diet using a metabarcoding approach across a wide urban–natural landscape gradient in the mid-Atlantic region. Concordant with our expectations and previous Big Brown Bat diet studies from visual identification, we observed a high abundance of Coleoptera (beetles) relative to other insect orders. Although a possible improvement over visual techniques for studying food habits, we suggest caution in interpreting metabarcoding results in diet studies. We noted observations of environmental or contaminant taxa within these data, and designed a stringent filtering method that we used to eliminate these taxa, but that also removed previously documented prey taxa from our dataset.