BackgroundHepatitis B vaccination is the most important preventive measure against Hepatitis B viral (HBV) infection. Vaccination against HBV infection among healthcare workers is important because of their daily exposure to patients’ body fluids and the possible risk of transmission to other patients. Hence, this study assessed the risk of hepatitis B infection, vaccination status and associated factors among healthcare workers in six geopolitical zones of Nigeria.MethodsA nationwide cross-sectional study was conducted between January and June 2021 using electronic data capture techniques to enroll 857 healthcare workers (HCWs) in regular contact with patients and their samples through a multi-stage sampling method.ResultsThe participants’ mean (SD) age was 38.7 (8.0) years, and 453 (52.9%) were female. Each of the six geopolitical zones in Nigeria had a fair representation, with a range of 15.3 to 17.7% of the study population. The majority (83.8%) of healthcare workers in Nigeria knew that they were at an increased risk of being infected by their work. Also, 72.2% knew that if infected, there was a high risk of liver cancer in later life. Many participants (642 [74.9%]) responded that they consistently applied standard precautions, such as hand washing, gloves, and face masks while attending to patients. Three hundred and sixty (42.0%) participants were fully vaccinated. Of the 857 respondents, 248 (28.9%) did not receive any dose of the hepatitis B vaccine. Factors that were associated with not being vaccinated included age less than 25 [adjusted odds ratio (AOR) 4.796, 95% CI 1.119 to 20.547,p= 0.035], being a nurse (AOR 2.346, 95% CI 1.446 to 3.808,p= 0.010), being a health attendant (AOR 9.225, 95% CI 4.532 to 18.778,p= 0.010), and being a healthcare worker from the Southeast (AOR 2.152, 95% CI 1.186 to 3.904,p= 0.012) in Nigeria.ConclusionThis study showed a high level of awareness of the risks associated with hepatitis B infection and suboptimal uptake of the hepatitis B vaccine among healthcare workers in Nigeria.