Background: Infection with hepatitis B virus (HBV) is a major worldwide health problem. The estimated prevalence of HBV is about 1.4% in Egypt. This infection is one of the main occupational hazards in the field of health care workers (HCW). Objectives: This study aimed to assess the prevalence of HBV infection among HCWs in Suez Canal University Hospital, East Egypt and their hepatitis B vaccination coverage. Methodology: A cross sectional descriptive study was conducted, including 450 Health Care Workers (HCWs) with more than 6 months of job experience, selected through stratified random sampling by HCW category. A structured questionnaire was used to collect demographic parameters, history of occupational exposures and HBV vaccination status. Blood samples were screened for HBsAg, hepatitis B core antibody, and hepatitis B surface antibody titer by ELISA. Results: Our study sample included 66.7% aged < 30 years. 73.5% of study sample were fully vaccinated, 10.7% were unvaccinated. Only 2.7% of those who have ever been vaccinated had HBsAb testing. There was a significant relation (p<0.001) between occupation and vaccination rate being the highest among nurses and doctors (80%, 78% respectively). Also a significant relationship (p<0.001) was found with education level and age, while no relation was found between gender and vaccination rate. The prevalence of chronic HBV infection and HBcAb were 0.4, and 15.6% respectively. HBV immunity achieved either by healed infection, or evidence of natural boosting was found among 13%. Immunity after vaccination was found in 75%, while 10% were still susceptible to infection. Only 21.1% report injury to infection control office and had viral markers testing. Conclusion: Although the use of hepatitis B vaccine decreased the incidence of HBV infection in HCWs, still there is a proportion of healthcare workers are unvaccinated. Therefore, new policies are needed for HBV screening, vaccination, and serological response.