BACKGROUND adequate knowledge on hepatitis B virus (HBV) infection is important among healthcare workers (HCWs) as this impacts the vaccination seeking behaviour. This study sought to assess the knowledge, vaccination status and related factors amongst HCWs in a tertiary facility in Ghana. METHODS an analytical cross-sectional study was conducted amongst full-time HCWs of different categories at the Cape Coast Teaching Hospital, Ghana. Stratified sampling was used to arrive at the number needed for each category of HCW and then simple random sampling to recruit participants. A structured self-administered questionnaire was used. Descriptive statistics and logistics regression were carried out on the data. RESULTS a total of 303 HCWs participated with 78.07% (n=235) being between 20-30 years, and majority being females (62.38%, n=189). A total of 186 (61.39%) participants had adequate knowledge, mean knowledge score was 4.73/7 (±0.97). About 80% (n=218) had received the 3 doses of HBV vaccine. Among the unvaccinated, cost was the major barrier (62.07%, n=18). Participants who did not know that HBV was more infectious than HIV (aOR=5.31, 95%CI: 1.91-14.77), p<0.001) and those who did not have knowledge that HBV vaccine was effective were more likely to be unvaccinated (aOR=8.63, 95%CI: 2.99-24.94), p<0.0001). The gender and cadre of staff did not show statistical evidence of an association with vaccination status. CONCLUSIONS knowledge on HBV is paramount for all HCWs as well as the importance of receiving the full doses of the hepatitis B vaccines. Barriers to vaccination must be removed to ensure protection of HCWs.