Human African trypanosomiasis became a neglected disease after the 1960s, when case numbers dropped dramatically. It again became a public health problem in sub-Saharan Africa at the end of the 1990s, when new cases were reported, notably in Central Africa, and specifically in Gabon, where historic foci existed and new cases have been reported. Therefore, the present study reports on an entomological survey conducted in May 2012 to determine the pathogenic trypanosome infection rate in tsetse flies and characterize the diversity of Trypanosoma species in the Ivindo National Park (INP) in northeastern Gabon. Nine Vavoua traps were used to catch tsetse over a 7-days period. All tsetse flies captured were identified to species, dissected, and trypanosome species identified using polymerase chain reaction (PCR). In total, 160 tsetse flies were analyzed, including Glossina palpalis palpalis, Glossina fusca congolense, and Glossina tachinoïdes. The trypanosome infection rate of the flies was 6.3 and 31.9% using microscopy and PCR, respectively. The species identified were Trypanosoma congolense savannah type, Trypanosoma brucei brucei, Trypanosoma brucei gambiense, Trypanosoma vivax, and Trypanosoma congolense forest type. Trypanosoma risk index was 0.75 and 7.05 for humans and for animals, respectively. This study illustrates the diversity of Trypanosoma species infecting the tsetse flies in the INP. The simultaneous occurrence of Trypanosoma and tsetse from the palpalis group may suggest that the reservoirs of African animal trypanosomiasis should be carefully monitored in this area.