BACKGROUND Toxoplasma gondii infection can lead to severe disease outcomes in immune-compromised people. This study sought to determine the seroprevalence of anti-T. gondii antibodies among pregnant women, hospitalized children (<5 years old) and HIV-seropositive persons in Accra. METHODS A cross-sectional study was conducted in two hospitals in Accra, and a total of 450 voluntary participants were recruited for the study consisting of 125 pregnant women, 200 children and 125 HIV-seropositive persons. Serum was obtained from venous blood safely drawn from each participant and tested for specific anti-Toxoplasma antibodies IgG and IgM by ELISA. A serological criterion for seropositivity was a positive test result for any of the two anti-Toxoplasma antibodies or a combination of both. Questionnaire interviews were conducted to obtain personal information and Toxoplasma infection risk-related data. RESULTS Those who tested seropositive for anti-T. gondii antibodies were 51.2 % (64/125) pregnant women, 58.0 % (116/200) children and 57.6 % (72/125) HIV patients. The major risk factors associated with anti-T. gondii seropositivity were identified as age (in children), handling raw meat and gravida status (in pregnant women). The results of this study confirmed that the seroprevalence of T. gondii infection is high among pregnant women, hospitalized children <5 years old and HIV patients. CONCLUSIONS A further study to investigate pre-pregnancy infections with T. gondii among women of childbearing age, seroconversion rate in pregnant women, rate of mother-to-child transmission and reactivated infections among HIV-seropositive persons is recommended.