Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and marsupials play an important role in the epidemiology of T. gondii because they are sources of infection for domestic and feral cats. Serum samples from 151 rodents and 48 marsupials, captured in the Atlantic Forest, São Paulo State, southeastern Brazil, were analyzed for the presence of T. gondii antibodies. Antibodies detected by the modified agglutination test (MAT ≥ 25) were found in 8.6% (13/151) of the rodents and 10.4% (5/48) of the marsupials, with titers ranging from 25 to 6400 and from 25 to 3200, respectively for the rodents and marsupials. Three of the eight species of rodents (Akodon spp., Oligoryzomys nigripesand Rattus norvegicus), and one from the four marsupial species (Didelphis aurita) presented positive animals. T. gondii was described for the first time in the rodent Oligoryzomys nigripes.