Bats are the second most diversified order of mammals. Most are nocturnal and highly-elusive, forming communities of species with cryptic morphologies and similar ecologies. The advent of molecular techniques allows us to apprehend many aspects of their biology. In Western Europe for instance, basic genetic tools only recently revealed that three species of long-eared bats (genus Plecotus) coexist. However, their field recognition remained complicated as external measurements and other morphological characters are overlapping among species. A first contribution of this work (chapter 2) concerned the development of new genetic tools alongside with the revision of morphological characters and distribution of species in Switzerland. The combined use of both nuclear and mitochondrial markers (chapter 3) on a geographically broad sampling showed that no hybridization occurred among species, which mate assortatively, but also have different genetic structuring potentially explained by different dispersal abilities and population dynamics. Finally, chapters 4 and 5 focused on the analysis of the fine-scale diet of the three bat species in a small area of sympatry around Geneva. To this end, COI-metabarcoding was conducted on faeces collected under maternity roosts across most of the active season, from April to October, causing minimal disturbance to these sensitive animals. While seasonal changes in insect composition explained most of the diet variation, the ecology of over 680 prey species identified also showed that predator species exploited different feeding habitats. Altogether, these various analyses provide new and robust information about the ecological mechanisms allowing for the stable coexistence of these closely-related species.