AbstractBats are well known for species richness and ecological diversity, and thus, they provide a good opportunity to study relationships and interaction between species. To assess interactions, we consider distinct traits that are probably to be triggered by niche shape and evolutionary processes. We present data on the trophic niche differentiation between two sympatric European trawling bat species, Myotis dasycneme and Myotis daubentonii, incorporating a wide spectrum of methodological approaches. We measure morphological traits involved in foraging and prey handling performance including bite force, weightlifting capacity and wing morphology. We then measure resulting prey consumption using both morphological and molecular diet analyses. These species closely resemble each other in morphological traits, however, subtle but significant differences were apparent in bite force and lift capacity, which are related to differences in basic body and head size. Both morphological and molecular diet analyses show strong niche overlap. We detected subtle differences in less frequent prey items, as well as differences in the exploitation of terrestrial and aquatic‐based prey groups. Myotis dasycneme feeds more on aquatic prey, like Chironomidae and their pupal stages, or on the aquatic moth Acentria ephemerella. Myotis daubentonii feeds more on terrestrial prey, like Brachycera, or Coleoptera. This suggests that these bats use different microhabitats within the habitat where they co‐occur.