Blastocystis sp. is a common intestinal parasite infecting humans and a wide range of animals worldwide. It exhibits an extensive genetic diversity and 17 subtypes (STs) have thus far been identified in mammalian and avian hosts. Since several STs are common to humans and animals, it was proposed that a proportion of human infections may result from zoonotic transmission. However, the contribution of each animal source to human infection remains to be clarified. Therefore, the aim of this study was to expand our knowledge of the epidemiology and host specificity of this parasite by performing the largest epidemiological survey ever conducted in animal groups in terms of numbers of species screened. A total of 307 stool samples from 161 mammalian and non-mammalian species in two French zoos were screened by real-time PCR for the presence of Blastocystis sp. Overall, 32.2% of the animal samples and 37.9% of the species tested were shown to be infected with the parasite. A total of 111 animal Blastocystis sp. isolates were subtyped, and 11 of the 17 mammalian and avian STs as well as additional STs previously identified in reptiles and insects were found with a varying prevalence according to animal groups. These data were combined with those obtained from previous surveys to evaluate the potential risk of zoonotic transmission of Blastocystis sp. through the comparison of ST distribution between human and animal hosts. This suggests that non-human primates, artiodactyls and birds may serve as reservoirs for human infection, especially in animal handlers. In contrast, other mammals such as carnivores, and non-mammalian groups including reptiles and insects, do not seem to represent significant sources of Blastocystis sp. infection in humans. In further studies, more intensive sampling and screening of potential new animal hosts will reinforce these statements and expand our understanding of the circulation of Blastocystis sp. in animal and human populations.