Didelphis (Marsupialia, Didelphimorphia) are synanthropic mammals, whose omnivorous diet predisposes them to infections caused by endoparasites. Their higher frequency in urban areas makes them potential carriers of zoonotic protozoans and helminths, enhancing potential transmission to humans. Our purpose was to study two common species, Didelphis albiventris (54 individuals) and D. aurita (2 individuals), which were screened for blood, skin and intestinal parasites in animals captured in urban areas and in riparian forest regions associated with the Capivari River Basin, in Monte Mor's municipality, São Paulo state (SP), Brazil. Blood and tissue samples were collected for DNA extraction and PCR. Fecal samples were collected and submitted to two sedimentation and two flotation methods. 77.6% of fecal samples were positive for nematode eggs, 34.5% for trematode eggs and 32.7% for protozoans. Two D. aurita specimens were naturally infected by Trypanosoma cruzi. Molecular analysis in a D. albiventris captured on a forested rural area was positive for Leishmania sp. DNA. Several parasites were found infecting Didelphis sp., demonstrating that this group of animals can harbor important zoonotic parasites, potentially playing a role as sylvatic reservoirs for human and domestic animal pathogens.