Blastocystis is an enteric protist and one of the most frequently reported parasitic infections in humans and a variety of animal hosts. It has also been reported in numerous parasite surveys of animals in zoological gardens and in particular in non-human primate species. PCR-based methods capable of the direct detection of Blastocystis in faeces were used to detect Blastocystis from various hosts, including non-human primates, Australian native fauna, elephants and giraffes, as well as their keepers from a Western Australian zoo. Additional faecal samples were also collected from elephants and giraffes from four other zoos in Amsterdam (The Netherlands), Antwerp (Belgium), Melbourne and Werribee (Australia). Information regarding the general health and lifestyle of the human volunteers were obtained by questionnaire. Overall, 42% and 63% of animals and zoo-keepers sampled from the Western Australian zoo were positive for Blastocystis, respectively. The occurrence of Blastocystis in elephants and giraffes from other cities was similar. This is the first report of Blastocystis found in the elephant, giraffe, quokka, southern hairy nosed wombat and western grey kangaroo. Three novel and what appear to be highly host-specific subtypes (STs) of Blastocystis in the elephant, giraffe and quokka are also described. These findings indicate that further exploration of the genetic diversity of Blastocystis is crucial. Most zookeepers at the Perth Zoo were harbouring Blastocystis. Four of these zoo-keeper isolates were identical to the isolates from the southern hairy nosed wombat and five primate species.