Background: Health Care workers (HCWs) have an approximate four-fold increased risk of contracting hepatitis B infection than the normal population. A lack of knowledge and practices regarding precautions has been frequently observed. We aimed to do a knowledge, attitude, and practices (KAP) study regarding hepatitis B prevention measures among HCWs.Methods: The study enrolled 250 HCWs. A questionnaire-based proforma regarding KAP towards hepatitis B, its causation, and prevention was filled out by each participant.Results: Mean age (SD) of participants was 31.8 ± 9.1 years with 83 males and 167 females. Subjects were divided into two groups: Group I (House Surgeons and Residents) and Group II (Nursing staff, Laboratory Technicians, Operation Theatre Assistants). All Group I and 148 (96.7%) of Group II subjects had adequate knowledge regarding the professional risk of hepatitis B virus transmission. Knowledge regarding different modes of transmission was less in Group II subjects (Blood (96.1%), Sex (84.3%), percutaneous route (85.6%), and During Birth (83%)) as compared to Group I (100% in all). All subjects in Group I and 134 (90.9%) Group II subjects knew about vaccination as a preventive measure. There was a slight discordance between attitude and practices towards universal precautionary measures in Group II subjects (Use of gloves 96.1% vs 94.8%; Safe needle disposal 96.7% vs 96.1%; Vaccination 94.8% vs 67.9%). Of the subjects in Group I, 94.8% were vaccinated and 67.9% were vaccinated in Group II, with complete vaccination rates being 76.3% and 43.1%, respectively, and the difference was statistically significant (P < 0.001).Conclusion: Better knowledge and attitude led to more adoption of preventive practices. But, still, there is a gap in the KAP towards hepatitis B preventive practices and knowledge is not getting transformed into practices. We recommend that all HCWs should be questioned about their vaccination status. In addition, vaccination coverage, various preventive campaigns, and the hospital infection control committee (HICC) need to be strengthened.