Myotis emarginatus is one of the few bats known to feed mostly on spiders. In order to study the importance of this type of prey, we analysed the species’ diet in five colonies across the Iberian Peninsula using amplicon metabarcoding in order to describe its composition at the species level, and analyse its geographic variability within the peninsula. We identified 138 prey species, belonging to 11 different arthropod orders. Among them, 45 species of spiders were identified, mostly of the orb-web building guild, as consumed by 82 out of 106 studied bats, corresponding to every colony and season sampled. Besides, lepidopterans and dipterans were also consumed in every colony. Among the latter, the stable fly Stomoxys calcitrans was especially important in two of the colonies, showing that M. emarginatus can also opportunistically exploit different resources or foraging grounds, such as cattle sheds, which affects the composition of its diet also at ordinal level of prey.