This survey aimed to estimate the prevalence of anti-Toxoplasma IgG and IgM antibodies in people living in north west Tuscany (central Italy) and to investigate the adherence to antenatal screening programs and access to the Toxo-test as well. Sera from a large sample of individuals suspected to have acute infection or from pregnant women (10,352 subjects) aged between 1 day and over 70 years were analysed for both IgG and IgM anti-Toxoplasma antibodies using an immunoenzymatic method or a chemo-luminescent immunoassay. Overall, the seroprevalence of IgG antibodies was 21.4% (95% CI 20.62-22.20). A positive trend according to age was found, with low positivity observed in younger age groups. Among women of reproductive age the prevalence of IgG antibodies was 19.4% (95% CI 18.64-20.26). The overall IgM seroprevalence was 1.07% (95% CI 0.87-1.27). A low IgM prevalence was also observed in women of reproductive age (0.8%; 95% CI 0.65-1.03). Our study seems to indicate that primary prevention is widespread among women. However, an epidemiological surveillance system for toxoplasmosis should be implemented, to assess the risk of congenital toxoplasmosis and to determine the true burden of disease in adults.