This study aimed to estimate dynamic changes in seroprevalence of Toxoplasma gondii within the general population living in the northern part of the Republic of Serbia (Province of Vojvodina) during a 14-year period. The differences in prevalence of anti-toxoplasma antibodies were analyzed in correlation with age, gender, residential area (rural/urban) and meteorological factors. In this cohort retrospective study, 24,440 subjects between 1 and 88 years old were enrolled. To determine the presence of T. gondii-specific IgM and IgG antibodies in serum samples, commercially available ELISA kits were used (Euroimmun, Luebeck, Germany). During the study period, the overall T. gondii seroprevalence was 23.5%. The seroprevalence continuously decreased over time from 31.7% in 2008 to 20.4% in 2021 (0.81% per year, p < 0.001). Approximately 2% of patients had a serologic profile positive for both anti-Toxoplasma IgG and IgM antibodies. The seroprevalence was higher (28.87%) among men compared to women (24.28%), while urban residents (24.94%) had lower seroprevalence than the rural population (28.17%). A statistically significant negative correlation (r = -0.559) was found between serologic profile of patients positive for both T. gondii IgG and IgM antibodies and the annual mean air temperature. No significant association was observed between seropositivity to T. gondii infection and examined meteorological factors. These data could be useful to national and regional health authorities to create an optimal health policy to reduce rate of T. gondii infections.