Global evidence suggests that hepatitis B viral (HBV) infection is endemic in Africa and perinatal transmission remains one of the most important modes of HBV transmission in this area. This cross-sectional survey examined the seroprevalence and knowledge of hepatitis B among pregnant women attending antenatal clinic (ANC) in a mission hospital in Ghana. Systematic sampling technique was used to recruit 196 pregnant women. The level of statistical significance was set at 0.05 alpha level. The hepatitis B prevalence estimate (hepatitis B surface antigen) was 10.2% (N = 20) and all of the participants were aware of HBV infection. Majority cited media (radio) as their main source of information. Approximately 86% of the participants (N = 168) associated HBV infection with a curse and 88.8% (N = 174) indicated witches and wizards as possible causes of the infection. Those with higher level of school education had high hepatitis B knowledge score (P < 0.01). Implementation of a health education program on the route of hepatitis B transmission is required in the study setting. Also, inclusion of hepatitis B education as part of ANC activities will enable HBV-positive pregnant women to appreciate the need for hepatitis B vaccination of their newborns at birth.