Understanding the trophic structure of species assemblages is crucial in order to comprehend how syntropic species coexist in space and time. Bats are the second most taxonomically diverse group of mammals and display a wide range of dietary strategies. Due to their ability to disperse over water, ca. 60% of all extant bat species occur on islands and for the most part their interspecific ecological interactions are poorly known. Using DNA metabarcoding, this study offers the first insights into the diet of Macaronesian bats by providing a holistic overview of prey consumed by all 3 bat species found on Madeira Island (Pipistrellus maderensis, Nyctalus leisleri verrucosus, and Plecotus austriacus) and investigating both interspecific (between P. maderensis and N. l. verrucosus) and intraspecific (between female and male N. l. verrucosus) dietary differences. We identified a total of 110 species of arthropod prey in the diet of the 3 bat species, including multiple agriculture and forestry pest species, a human disease-relevant species, and numerous taxa not previously recorded on the island. Lepidoptera was the primary prey order for all 3 bat species. The diet composition of P. maderensis and N. l. verrucosus differed significantly, with P. maderensis consuming more Diptera and multiple prey taxa not found in the diet of N. l. verucosus. Moreover, male N. l. verrucosus exhibited a broader niche breadth than females. This study is among the first to use DNA metabarcoding to evaluate the diet of insular bats and thus greatly advances knowledge regarding the trophic ecology and pest suppression services of these poorly-known mammals.