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Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) were captured in the course of three seasons (2017–2020) in the Zoo Košice. The total number of captured individuals was 5,667 biting midges, and 12 species of Culicoides were identified, while the most frequently captured and the most populous species were C. obsoletus/C. scoticus, C. furcillatus, and C. festivipennis. The most frequent hosts on which the biting midges were blood-feeding were humans (Homo sapiens), Burchell's zebra (Equus quagga burchellii), and domestic cow (Bos taurus). This is the first study to confirm a wide range of biting midges species in a zoo which are capable of using not only animals as their hosts, but also humans, in whom a bite may induce an allergic reaction. More than 80% of the fauna of biting midges in the Zoo Košice represented Culicoides obsoletus/C. scoticus, which are in the Central Europe regarded as the potential vectors of arboviruses, to which primarily ruminants are highly susceptible.