To investigate the possible role of common brushtail possums (Trichosurus vulpecula) in the transmission of Toxoplasma gondii within a zoo environment, a serological survey of a free-ranging population resident within Taronga Zoo, Sydney, Australia was undertaken using the modified agglutination test (MAT). For comparison, the seroprevalence of T. gondii antibodies was also assessed in a possum population inhabiting a felid-free, non-urban woodland habitat. Six of 126 possums (4.8%) from the zoo population had antibodies to T. gondii with a MAT titre of 25 or higher, while in contrast, all of the 17 possums from woodland were seronegative. These observations suggest that possums were at a higher risk of exposure to the parasite as a consequence of co-existing with domestic, stray and captive felids associated with urbanisation. Screening of captive felids at the zoo indicated 16 of 23 individuals (67%) and all 6 species were seropositive for T. gondii, implicating them as a possible source of the parasite within the zoo setting. In addition captive, non-felid carnivores including the chimpanzee (Pan troglodytes), saltwater crocodile (Crocodylus porosus), dingo (Canis lupis) and leopard seal (Hydrurga leptonyx) were tested for the presence of T. gondii antibodies as these species predate and are a leading cause of death amongst zoo possums. In total, 5 of 23 individuals (22%) were seropositive, representing 2 of the 4 carnivorous species; the dingo and chimpanzee. These data suggest that carnivory was not a highly efficient pathway for the transmission of T. gondii and the free-ranging possum population posed minimal threat to the health of zoo animals.