Background: Toxoplasma gondii, a zoonotic parasite, is one of the wide spread parasitic causes of asymptomatic infection in human, yet can cause severe disease and disorders in infants, when infected during pregnancy, and also in immunocompromised individuals. Thus, updated information about the prevalence of the infection in each region and time period is necessary. Objectives: The aim of the present study was to determine the prevalence of the infection in patients referred to Dr. Sharifi clinical laboratory of Isfahan, Iran, during January 2014 to January 2015. Methods: In a retrospective study, 1287 patients that had anti-Toxoplasma IgM and/or IgG test from January 2013 to January 2015 were selected and included in the study. Quantitative determination of anti-Toxoplasma IgM and IgG was performed using antibody capture chemiluminescence immunoassay (CLIA) kits (LIAISON® Toxo IgM and IgG, DiaSorin S.P.A, Italy) applied using the LIAISON (DiaSorin, Germany) device. All the available variables such as anti-Toxoplasma IgG and IgM concentrations, gender and age were recorded and analyzed. Results: Overall, 1287 cases including 1215 (94.4%) females and 72 (5.6%) males with mean age of 28.64 years (min: 1, max: 78) were studied. The results showed that 36 (2.8%) out of 888 and 325 (25.3%) out of 1243 studied individuals were anti-Toxoplasma IgM and IgG seropositive, respectively. The mean age observed significantly higher in IgG positive humans (P < 0.001), but not in IgM positive ones (P = 0.065). No statistically significant relationship was observed for the IgM and IgG seropositivity and concentrations among the genders. Conclusions: According to the results of the present study, prevalence of infection with T. gondii is high in Isfahan, yet it is still lower than most of the other studied regions in the country. Also, the risk of the infection rises with increasing age.