Abstract: Mutton signifies one of the most prevalent sources for human toxoplasmosis. However, sheep serological assays don't categorize the virulent strains initiating antibodies, so the biological bioassay of Egyptian mutton isolates with reference to their pathogenicity in both mice and kittens were done in this study for indicating to how extent their zoonotic bio-hazard. A total number of 280 of each sheep blood and tissue samples were collected during slaughtering at Cairo abattoir, Egypt. Sera assayed using Latex Agglutination Test (LAT) and immunosorbant assay (ELISA) and their corresponding mutton samples were microscopically examined after pepsin digestion for detection of Toxoplasma gondii infection. The sero-positive percent of the naturally infected sheep was 50.4 and 61.4 by LAT and ELISA, respectively, 47.9% of samples were confirmedly positive in both LAT and ELISA results. The microscopical examination revealed that only 28 out of 134 (20.9%) of the confirmed sero-positive animals by both tests were found harboring T. gondii tissue cysts in their mutton samples, while high percentage of confirmed sero-positve animals (79.1%) (106 out of 134) were biologically tissue cysts free mutton. Biological typing of the 28 T. gondii sheep isolates with reference to mice and kittens' bioassay indicated that 10.7, 50, 21.4 and 17.9% were type I, II, III and avirulent strains, respectively. The high T. gondii infection rate resulted in this study concludes that the feeding of under cooked mutton is a bad health habit as a source for human toxoplasmosis moreover; the T. gondii virulent strains obtained by mutton bioassay indicated that not all sero-positive sheep are connecting zoonotic bio-hazard through their mutton strains.