Blastocystis is an anaerobic protist, commonly inhabiting the intestinal tract of both humans and other animals. Blastocystis is extremely diverse comprising 17 genetically distinct subtypes in mammals and birds. Pathogenicity of this enteric microbe is currently disputed and knowledge regarding its distribution, diversity and zoonotic potential is fragmentary. Most research has focused on Blastocystis from primates, while sampling from other animals remains limited. Herein, we investigated the prevalence and distribution of Blastocystis in animals held within a conservation park in South East England. A total of 118 samples were collected from 27 vertebrate species. The barcoding region of the small-subunit ribosomal RNA was used for molecular identification and subtyping. Forty one per cent of the species were sequence positive for Blastocystis indicating a high prevalence and wide distribution among the animals in the park. Six subtypes were identified, one of which is potentially novel. Moreover, the majority of animals were asymptomatic carriers, suggesting that Blastocystis is not pathogenic in animals. This study provides a thorough investigation of Blastocystis prevalence within a wildlife park in the UK and can be used as a platform for further investigations on the distribution of other eukaryotic gut microbes.