Toxoplasma gondii is a protozoan parasite that can be transmitted to humans through a variety of routes including blood transfusion. This study aimed to investigate the seroprevalence of T. gondii infection and associated risk factors in healthy blood donors in Tunisia. A total of 800 healthy blood donors from two blood centers in south and coastal Tunisia were analyzed for anti-T. gondii IgG and IgM antibodies by indirect immunofluorescence assay (IFA) and enzyme-linked immunoassays (ELISA), respectively. Structured questionnaires were used to gather information on risk factors for T. gondii infection during collection. The overall seroprevalence was 44.4% of which 352 (44%) and 3 (0.4%) were positive for IgG and both IgG and IgM anti-T. gondii antibodies, respectively. Multivariate analysis showed that T. gondii seropositivity was significantly associated with the birth place (adjusted odds ratio [OR] = 2.72; 95% confidence interval [CI]: 1.49–4.94) and the age of the donors (adjusted OR = 4.98; 95% CI: 1.50–16.58) which are independent risk factors. In addition, the variables of hand washing before eating (adjusted OR = 0.52; 95% CI: 0.37–0.74) and living in an urban environment (adjusted OR = 0.30; 95% CI: 0.13–0.71) are two protective factors. This study provided the first data on the seroprevalence and epidemiology of T. gondii infection in healthy blood donors in Tunisia.