The gut microbiome is essential for a host to digest food, maintain health, and adapt to environments. Bacterial communities of gut microbiota are influenced by diverse factors including host physiology and the environment. Many non-human primates (NHPs), which are physiologically close to humans, are in danger of extinction. In this study, the community structure of the gut microbiota in three NHPs: siamangs (Symphalangus syndactylus, Ss), Bornean orangutans (Pongo pygmaeus, Pp), and white-handed gibbons (Hylobates lar, Hl)—housed at the largest Zoo in Taiwan were analyzed. Pp and Ss were housed in the Asian tropical rainforest area, while Hl was housed in two separate areas, the Asian tropical rainforest area and the conservation area. Bacterial community diversity of Ss, indicated by the Shannon index, was significantly higher compared with that of Hl and Pp, while the richness (Chao 1) and observed operational taxonomic units (OTUs) were similar across the three species of NHPs. Host species was the dominant factor shaping the gut microbial community structure. Beta-diversity analysis including non-metric multidimensional scaling (NMDS) and unweighted pair group method with arithmetic mean (UPGMA) suggested gut bacterial communities of Hl housed in the conservation area were closely related to each other, while the bacterial communities of Hl in the rainforest area were dispersedly positioned. Further analysis revealed significantly higher abundances of Lactobacillus fermentum, L. murinus, and an unclassified species of Lactobacillus, and a lower abundance of Escherichia-Shigella in Hl from the conservation area relative to the rainforest area. The ratio of Lactobacillus to Escherichia-Shigella was 489.35 and 0.013 in Hl inhabiting the conservation and rainforest areas, respectively. High abundances of Lactobacillus and Bifidobacterium and a high ratio of Lactobacillus to Escherichia-Shigella were also observed in one siamang with notable longevity of 53 years. Data from the study reveal that host species acted as the fundamental driving factor in modulating the community structure of gut microbiota, but that habitats also acted as key determinants within species. The presence and high abundance of probiotics, such as Bifidobacterium and Lactobacillus, provide potential indicators for future diet and habitat optimization for NHPs, especially in zoological settings.