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In view of the worldwide importance of Toxoplasma gondii and Neospora caninum and the limited data on the seroprevalence of these parasites in Egypt, this study aimed to estimate the prevalence of anti-T. gondii and anti-N. caninum antibodies in rabbits, cattle, and humans. We used ELISA methods based on surface antigen 2 of T. gondii (TgSAG2t) and surface antigen 1 of N. caninum (NcSAG1t). High seroprevalence of T. gondii (51.49%) was detected in pregnant women, and antibodies to N. caninum were also detected in human samples (7.92%). Anti-T. gondii or N. caninum antibodies were detected in cattle (TgSAG2t: 10.75%; NcSAG1t: 20.43%). In rabbits, only one sample was N. caninum positive (1.85%). The high prevalence of toxoplasmosis and neosporosis in cattle affects the development of the livestock industry and is also an important infective source for human infection in Egypt.