Toxoplasmosis is a global zoonotic disease that poses a threat to both animal and human, therefore the present investigation's aims were to detect T. gondii antibodies and T. gondii DNA in sheep and goat milk samples, and the relationship between epidemiological aspects and T. gondii infection. The current study was carried out on lactating sheep and goats (50 of each) from farmers’ houses (El-Fateh and Abnoub) in Assiut Governorate. CMT was performed on milk samples to detect subclinical mastitis, while LAT and PCR were utilized to diagnose T. gondii. Among the 100 examined animals, 2 (2%) were classified as CMT (++), 9 (9%) as CMT (+), 13 (13%) as suspicious and 76 (76%) as negative. According to LAT, 26% (13/50) of sheep milk samples and 44% (22/50) of goat milk samples contained T. gondii antibodies. By using PCR, it was discovered that 86% (43/50) of dairy sheep and 94% (47/50) of dairy goats had T. gondii DNA in their milk. The T. gondii infection in PCR-examined dairy sheep varied significantly by location (P<0.01), but there was no discernible change based on age (P<0.05). The percentages of molecularly testing dairy goats infected with T. gondii did not significantly differ (P<0.05) based on their locality and age. In order to stop and limit the spread of toxoplasmosis in Assiut Governorate, surveillance and sufficient biosecurity measures must be implemented