Mountain gorillas (Gorilla beringei beringei) are critically endangered primates surviving in two isolated populations in protected areas within the Virunga Massif of Rwanda, Uganda, the Democratic Republic of Congo, and in Bwindi Impenetrable National Park in Uganda. Mountain gorillas face intense ecologic pressures due to their proximity to humans. Human communities outside the national parks, and numerous human activities within the national parks (including research, tourism, illegal hunting, and anti-poaching patrols), lead to a high degree of contact between mountain gorillas and wildlife, domestic animals, and humans. To assess the pathogen transmission potential between wildlife and livestock, feces of mountain gorillas, forest buffalo (Syncerus caffer nanus), and domestic cattle (Bos taurus) in Rwanda were examined for the parasites Giardia and Cryptosporidium. Giardia was found in 9% of mountain gorillas, 6% of cattle, and 2% of forest buffalo. Our study represents the first report of Giardia prevalence in forest buffalo. Cryptosporidium-like particles were also observed in all three species. Molecular characterization of Giardia isolates identified zoonotic genotype assemblage B in the gorilla samples and assemblage E in the cattle samples. Significant spatial clustering of Giardia-positive samples was observed in one sector of the park. Although we did not find evidence for transmission of protozoa from forest buffalo to mountain gorillas, the genotypes of Giardia samples isolated from gorillas have been reported in humans, suggesting that the importance of humans in this ecosystem should be more closely evaluated.