Myotis emarginatus seems fond of spiders and flies, a unique feeding style among European bats. The importance of each prey type varies among studies, so this paper aims to expand on the knowledge to unveil the trophic niche of M. emarginatus by studying its diet changes throughout the maternity season. We sampled five maternity colonies in the Basque Country every 2 weeks for the whole duration of the maternity season and studied their diet using DNA metabarcoding methods. We observed significant changes in diet diversity and composition. At the beginning of the season, M. emarginatus consumed a variety of prey orders, Diptera being the most abundant; but as the season progressed, the relative consumption of Araneae increased. By August, 80% of the weighted percentage of occurrences (wPOO) corresponded to the orb-web-building spider Araneus diadematus. Orb-web-building spiders need habitats of high vertical complexity to grow in abundance. Therefore, conserving them is essential to preserve attractive hunting grounds for colonies of M. emarginatus. Besides, diurnal flies, mainly Stomoxys calcitrans, were also considerably consumed in the colonies studied, which can become detrimental to the well-being of cattle, and can act as a vector of many diseases.