Enterocytozoon bieneusi, a fungus-like protist parasite, causes symptomatic and asymptomatic intestinal infections in terrestrial animals and is also abundant in the environment. This parasite has been isolated from a variety of host types including humans, livestock, companion animals, birds, and wildlife, as well as the natural and urban environments including drinking source water, coastal water, recreational water, wastewater, vegetables in retail markets, and raw milk on farms. E. bieneusi exhibits high genetic diversity among host species and environmental sources and at least 500 genotypes have been identified thus far. Since its discovery in AIDS patients in 1985, scientists across the world have worked to demonstrate the natural history and public health potential of this pathogen. Here we review molecular typing studies on E. bieneusi and summarize relevant data to identify the potential sources of human and nonhuman infections and environmental contamination. This review also discusses the possible transmission routes of E. bieneusi and the associated risk factors, and advocates the importance of the One Health approach to tackle E. bieneusi infections.