Entamoeba histolytica is an enteric parasite that infects approximately 50 million people worldwide. Although E. histolytica is a zoonotic parasite that has the potential to infect nonhuman primates, such transmission is poorly understood. Consequently, this study examined whether E. histolytica is present among humans, chimpanzees and baboons living in the Greater Gombe Ecosystem (GGE), Tanzania. The primary aims were to determine patterns of E. histolytica infection in a system with human-nonhuman primate overlap and to test associations between infection status and potential risk factors of disease. Entamoeba spp. occurred in 60.3% of human, 65.6% of chimpanzee and 88.6% of baboon samples. Entamoeba histolytica occurred in 12.1% of human, 34.1% of chimpanzee and 10.9% of baboon samples. Human E. histolytica infection was associated with gastrointestinal symptoms. This was the first study to confirm the presence of E. histolytica in the GGE. The high sample prevalence of E. histolytica in three sympatric primates suggests that zoonotic transmission is possible and stresses the need for further phylogenetic studies. Interventions targeting better sanitation and hygiene practices for humans living in the GGE can help prevent E. histolytica infection in humans, while also protecting the endangered chimpanzees and other primates in this region.