We examined the prevalence of antibodies to zoonotic protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii, and Encephalitozoon cuniculi) and protozoans of veterinary importance (Neospora caninum, Sarcocystis neurona, and Besnoitia darlingi) in a population of North American opossums (Didelphis virginiana) from Louisiana. Samples from 30 opossums were collected as part of a survey for T. cruzi in Louisiana. Frozen sera from these 30 opossums were examined using an indirect immunofluorescent antibody test (IFAT) against in vitro-produced antigenic stages of these protozoans. Additionally, 24 of the 30 samples were examined using hemoculture, and all 30 were examined in the modified direct agglutination test (MAT) for antibodies to To. gondii. The prevalences of reactive IFAT samples were as follows: 60% for T. cruzi, 27% for To. gondii, 23% for E. cuniculi, 17% for S. neurona, 47% for B. darlingi, and 0% for N. caninum. Hemoculture revealed that 16 (67%) of 24 samples were positive for T. cruzi, compared to 18 of 30 (60%) by IFAT. The sensitivity and specificity for the IFAT compared to hemoculture was 100% for each. The modified direct agglutination test revealed that 9 (30%) of the 30 samples from opossums had antibodies to To. gondii, compared to 8 (27%) using the IFAT. The sensitivity and specificity of the IFAT compared to the MAT was 100% and 72%, respectively.