peer reviewed ; The diet of four maternity colonies of serotine bats in Southern Belgium was investigated by analysing faecal pellets collected from beneath the roost throughout the activity season. Their diet is composed of Coleoptera Melolonthidae (Melolontha sp., Amphimallon sp., Rhizotrogus sp., Serica brunnea), Coleoptera Scarabaeidae (Aphodius sp., Geotrupes sp.), Coleoptera Carabidae, Diptera Tipulidae, Diptera Chironomidae, Lepidoptera, Hemiptera Pentatomidae, Hymenoptera Ichneumonoidea Ophionidae, Trichoptera and Arachnida. The diet of an urban colony of serotine bats was broadly the same as the diets of three rural colonies. Though some qualitative and quantitative variations were observed between study sites, the main source of variation in the diet was the seasonal availability of potential prey. The prominence of agriculture-dependant prey (chafers in mid summer and Aphodius beetles in late summer and autumn) was observed at all study sites. Consequently, dietary breadth and diversity is smaller during these periods.