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Blastocystis spp. has been reported in wildlife, domestic animals and animals housed in ZOO. To-date, 17 genetically diverse lines have been reported in mammals and birds (designated ST) based on differences in the SSU rRNA. In this study, faeces samples were collected from 24 ZOO animals with clinical signs suggestive of gastrointestinal disease in Košice ZOO, Slovakia. After DNA isolation, PCR was conducted to amplify the SSU region of DNA of Blastocystis species. Forward primer- Blast F and reverse primer- Blast R were used in the reaction. From 25 faeces samples, Blastocystis spp. was detected in 5 animals (3 mammals, 2 birds), with a prevalence of 20%. Subsequent molecular analyses identified the ST 5 (n = 3), ST 7 (n = 1), and ST 12 (n = 1) subtypes, where the ST 5 subtype was identified in the mammalian group and birds, and the ST 7 and ST 12 subtypes were identified only in mammals. Based on these findings, focusing on ZOO animals as a potential source of infection for humans is highly recommended.