Toxoplasma gondii is a globally prevalent zoonotic parasite with major public health and veterinary impacts. In Egypt, One Health data on its distribution among blood donors and livestock are limited. This multicentric cross-sectional study aimed to determine the seroprevalence of Toxoplasma gondii among asymptomatic blood donors and livestock across six different Egyptian governorates and to identify the associated risk factors. Serum samples of 2000 asymptomatic blood donors and 500 animals (cattle and buffaloes) were tested for anti-Toxoplasma gondii IgG and IgM antibodies using ELISA. Sociodemographic and behavioral data were collected using a structured questionnaire. Overall IgG seroprevalence in blood donors was 41.8%, with IgM seropositivity at 2.1%. Significant regional variation was observed (p < 0.001), ranging from 66.5% in Alexandria to 16% in Cairo. The most significant risk factors were consumption of undercooked meat, contact with cats, and engagement in agricultural activities (p < 0.001). Among livestock, IgG and IgM seroprevalence were 7.2% and 5.2%, respectively, indicating active parasite circulation. The presence of seropositivity among apparently healthy donors raises concerns regarding transfusion safety. These findings highlight substantial and regionally heterogeneous Toxoplasma gondii exposure in Egypt. A One Health approach integrating public health education, food safety measures, and targeted screening of high-risk populations and animals is essential to reduce disease burden.