Background: Toxoplasma gondii is one of the parasitic protozoa with worldwide distribution that affects warm-blooded lives. Toxoplasmosis transmission does not occur from one-to-another, with the exception for transplacental route, blood transfusion and organ transplantation. Objectives: To measure the occurrence rate of Toxoplasma gondii infection among healthy blood donors and highlight the associated risk factors among this important group. Methods: Cross-sectional research was conducted in Menoufia University hospital blood transfusion center. Six hundred and thirty blood donors were recruited in this study. They filled a self-administered questionnaire including personal data and suspected risk factors as well as routine investigation for blood donors and serological tests for Toxoplasma gondii (IgG and IgM) were performed. Results: IgG sero-prevalence was 11.1%, IgM was 5.9%, both IgG and IgM positivity was 0.3% with total toxoplasmosis sero-prevalence 17%, Poor knowledge about toxoplasmosis transmission, dealing with cat, agricultural environment, eating ready to eat or undercooked meat, improper hand washing practices and AIDS infection were significantly associated with toxoplasmosis infection. Multivariate logistic regression analysis revealed that gender (AOR, 26.21; 95% CI: 7.77-88.45),poor knowledge (AOR, 15.39; 95% CI: 6.55-36.16), eating ready to eat meat(AOR, 50; 95% CI: 30-215), agricultural environment (AOR, 6.33; 95% CI: 3.07 – 13.05) and decreased frequency of hand washing (AOR, 30.66; 95% CI: 8.73-107.64)were independent risks for toxoplasmosis. Conclusion: Toxoplasma gondii positivity is prevalent among healthy blood donors. This finding underscores the importance of screening of toxoplasmosis among them and favors health education to improve the public knowledge about toxoplasmosis for proper prevention of infection.