Blastocystis spp. is an enteric protist that is present worldwide. Despite being discovered a century ago, there is still much to be learned about its pathogenicity and transmission. Different subtypes (ST) of Blastocystis spp. have been identified in various hosts, including humans, birds, and insects, and there is potential for zoonotic transmission through contact between humans and animals. The prevalence of Blastocystis spp. in humans and macaques in Singapore was understudied, and the findings revealed a significant prevalence of the parasite, with rates of 90% and 100% observed in each respective Macaca fascicularis population 1 and 2, with main subtypes (ST1, ST2, ST3, and ST5). Using metagenomics, the different subtypes of Blastocystis spp. (comprising ST2, ST3, and ST17) were identified in a healthy Singaporean cohort. Additionally, seven incidental findings of Blastocystis spp. were discovered in human patients with other gut parasites, including two ST1, two ST2, two ST3, and one ST8. Several factors such as diet or reverse zoonotic transmission are suggested to play a role in Blastocystis sp. subtype distribution.