Reserva Biológica de Duas Bocas (2,910 ha) is one of the largest Atlantic forest remnants in the State of Espírito Santo, Southeastern Brazil. We recorded non-volant tetrapods in this area from May 2007 through April 2008, using pitfalls, live traps, camera traps, and diurnal and nocturnal opportunistic searches. In addition, we compiled available museum and literature records from this area. We documented 52 species of amphibians, 24 species of non-avian reptiles, and 39 species of non-volant mammals. Out of these 115 species, 47 are new records for this area and six other species had their geographic ranges expanded with the present study. Furthermore, we present the record of predation of the tree frog Hypsiboas faber by the snake Chironius bicarinatus. Out of the species listed, five species are listed as threatened with extinction in the State of Espírito Santo, and many others have uncertain conservation status. Reserva Biológica de Duas Bocas is an important wildlife refuge, especially considering the expansion of urban areas in its surroundings.