Trypanosomes are known as widespread blood parasites of birds; however, knowledge of their prevalences in vectors and their overall biodiversity is rather limited. To assess the prevalences in potential vectors, we have microscopically examined ornithophilic bloodsucking Diptera (Culicidae, Simuliidae and Hippoboscidae) for the presence of trypanosomatids in their guts. In total, 3270 specimens were dissected, namely Culex pipiens Linnaeus, 1758 (n = 898), C. modestus Ficalbi, 1890 (136), Simulium vernum (Macquart, 1838) (1455), S. angustipes Edwards, 1915 (221) and Ornithomyia avicularia (Linnaeus, 1758) (560). All insect species were found to be infected with trypanosomatids, and the prevalence ranged from 4 to 8% but reached 60% in S. vernum. Blackflies and hippoboscids exclusively harboured trypanosomes (both T. cf. avium s.s. Danilewsky, 1885; T. corvi/culicavium group in hippoboscids). Mosquitoes were infected with T. culicavium Votypka, 2012 and T. avium s. l. but also with monoxenous parasites, namely Crithidia brevicula Frolov and Malysheva, 1989, and Paratrypanosoma confusum Votypka and Lukes, 2013. Only 4% of the isolated parasite strains were monoxenous whereas the majority were avian trypanosomes, confirming the vectorial status of the studied insects.