This report describes toxoplasmosis infection in a black-headed night monkey (Aotus nigriceps) from the Zoological Park of the Federal University of Mato Grosso, Brazil. The animal was submitted alive to the Veterinary Hospital of the Federal University of Mato Grosso presenting neurologic clinical signs. There were no improvements on the animal's clinical condition after treatment, so it was euthanized. At necropsy, the most significant gross lesions observed were hemorrhages diffusely scattered throughout the cerebral hemispheres. Histologic findings were characterized by nonsuppurative encephalitis and mild multifocal necrotizing nonsuppurative myocarditis. Groups of tachyzoites and cysts were observed surrounding the inflamed areas of cerebral parenchyma. Sections of the brain were submitted for immunohistochemistry examination for Toxoplasma gondii and Neospora caninum and also for polymerase chain reaction (PCR) to determine the presence of Toxoplasma gondii. The samples were positive for Toxoplasma gondii by PCR; however, cross reactivity was observed between Toxoplasma gondii and Neospora caninum antibodies. These findings were consistent with a toxoplasmosis infection in this animal.