Toxoplasmosis is an important infection caused by the single-celled parasite Toxoplasma gondii, which is a zoonotic parasite causing widespread human and animal diseases, mostly involving the central nervous system. Humans can acquire toxoplasmosis by ingestion of raw or undercooked meat containing T. gondii tissue cysts, ingestion of oocysts shed by infected felids via contaminated food or water, and by vertical transmission to the fetus through the placenta from the mother during pregnancy. The aim of the present study was to determine the seroprevalence of specific anti-T. gondii IgG and IgM antibodies using a large set of clinical diagnostic laboratory data obtained over a 14-year period. In total, 25 069 unique patients were included in the present study. The overall specific anti-T. gondii IgG prevalence were 36.3%, which was significantly (p < 0.01) higher than IgM prevalence (2.4%). Mean age for IgG antibody-positive patients was 33.7 ± 12.2 years. A significant positive correlation (r = 0.99; p < 0.01) was observed between age group and anti-T. gondii IgG antibody prevalence, which ranged from 4.2% to 66.7%. The most prevalent (69.9%; 95% CI 69.2–70.7) comorbidities of patients tested for presence of anti-T. gondii IgG and IgM antibodies were classified as factors affecting health status which includes also monitoring of normal pregnancy.