Chronic infection with hepatitis B virus (HBV) is a major public health concern in South Africa. Hepatitis B virus is a highly infectious blood-borne virus causing liver disease. Healthcare workers (HCWs) are at high risk of occupational exposure. This study aimed to investigate HBV vaccination amongst HCWs at a tertiary academic hospital in Gauteng province, South Africa. Self-administered questionnaires were used to collect data from 500 consecutively sampled HCWs. Data were analysed using Stata version 12. A total of 460 HCWs participated in the study. Most were women (68.7%), < 40 years of age (66.9%) and worked for < 10 years (66.0%). Almost 50.0% were either doctors or medical students and 40.3% were nurses or student nurses. Most HCWs in the age group of < 30 years (79.4%) had received at least 1 dose of HB vaccine. Prevaccination immunity screening was conducted on 17.5% of the HCWs, and only 11.0% reported to be protected against HBV. About 49.0% of HCWs were fully vaccinated. Post-vaccination immunity testing was conducted on 15.1%, and 24.0% of HCWs paid for vaccinations. Nursing staff and those with > 10 years of work experience were 2.5 and 2.6 times more likely to be vaccinated, respectively. Cleaning staff were less likely to be vaccinated. Although not all HCWs were fully vaccinated, our study found a higher proportion of fully vaccinated HCWs than previously reported in Gauteng Province. It is recommended that HB vaccination be promoted and a local vaccination policy, aligned with the national policy, be developed and implemented for all HCWs at the tertiary academic hospital.