Background Exposure to Toxoplasma gondii infection is prevalent in up to 90% of the world’s population. Severe infections can be fatal and deforming in neonates. Cats serve as reservoirs for T. gondii infections. Even though many Ghanaians have cats as pets in their homes only few studies have been undertaken to assess the prevalence of the disease in Ghana. The objective of the study was to determine the seroprevalence of T. gondii infection and its associated risk factors among pregnant women seeking antenatal care at the Goaso Municipal Hospital, in Asunafo North District of the Ahafo Region and the St. Michael’s Catholic Mission Hospital in the Bosomtwe District in the Ashanti Region of Ghana. Methods A cross-sectional study involving 266 (146 from Asunafo North District and 120 from Bosomtwe District) pregnant women who consented to participate in the study was undertaken. To gather information on associated risk factors, structured questionnaire was administered to the participants. In addition, 3 mls of venous blood was collected from each participant for anti-T. gondii IgG and anti-T. gondii IgM antibody analyses using ELISA. Results A total of 64.3% (171/266) of the study participant were positive for anti-T. gondii IgG and 26.3% (70/266) were positive for anti-T. gondii IgM antibodies. This signifies that 64.3% of the study population have been exposed to the infection and 26.3% were actively infected. There were no differences (P=0.383) between the overall T. gondii seroprevalences in the two Districts. There was a significant difference between antibody concentration levels and age in the Bosomtwe District in Ashanti Region. No association (OR