Introduction Toxoplasmosis is an acute infectious anthropozoonotic disease with mild asymptomatic clinical manifestations in immunocompetent persons and more severe in immunocompromised patients. Acute infection in pregnancy can result in severe congenital toxoplasmosis with severe sequels. Objective Aims of study were to detect Toxoplasma gondii seroprevalence in general population of Vojvodina, Serbia, differences between genders and determination of seroprevalence in women of reproductive age and pregnant women. Methods Our retrospective study was conducted from 2006 to 2008 including 625 immunocompetent patients, hospitalized or observed as outpatients at the Clinical Centre of Vojvodina, Novi Sad. We performed commercial ELISA kits SERION - ELISA classic test by VIRION for the presence of specific IgG and IgM antibodies. According to seroepidemiological aim of the study, our results were presented only in qualitative values. Results We observed 173 male and 452 female patients. Seroprevalence in general population of Vojvodina was 38.1%. In male population seroprevalence was 45.7%, and in female population it was 35.2%, the difference which was statistically significant (p<0.05). Seroprevalence increased with age and seroconversion was detected to occur in persons aged about 20 years. In all female patients, 353 (78.1%) were in reproductive age with seroprevalence of 30%. In 161 pregnant women seroprevalence was 31.7%. Conclusion In this study we screened actual seroepidemiological situation to Toxoplasma gondii in Vojvodina, thus giving a contribution to the continuous epidemiological screening done in this region and in the country. According to our results, almost 70% of women in reproductive age were sensitive to primary acute infection during further pregnancies, which is highly important for the prevention of congenital toxoplasmosis. Although not routinely conducted in many countries, routine serological testing to Toxoplasma gondii in pregnant women and their education about preventive measures against this infection could be an effective measure in the future.