Background and Objectives: Health Care Workers (HCWs) are at a high risk of needle stick injuries and HBV infection in Egypt; this problem is further aggravated by low Hepatitis B (HB) vaccination coverage. Limited data are available on the prevalence of HBV infection in Egyptian HCWs. In this study, we aimed to assess the HBV infection rate and genotypes among Egyptian HCWs. Methods: Five hundred and sixty-four (564) HCWs were included. Of them, 258 (45.74%) were health care providers and 306 (54.25%) were non-health care providers. All HCWs completed both the study questionnaires and provided a blood sample for HBV testing. Indeed, all HCWs were tested for Hepatitis B surface antigen (HBsAg) and antibody to Hepatitis B core antigen (anti-HBc), by enzyme-linked immunosorbent assay. HBVDNA was checked for HCWs who tested positive for HBsAg and/or anti-HBc, by nested Polymerase Chain Reaction (PCR). HBVDNA positive HCWs were further subjected to HBV genotyping. Results: The mean age of included HCWs was 33.0 ± 9.8 years, of whom 319 (56.56%) were males. The mean duration of health care work was 9.3 ± 6.7 years. The frequency of HBsAg and anti-HBc were 1.4%, and 24.5%, respectively. Old age and prolonged duration of health care work were significantly associated with anti-HBc seropositivity. Among 140 HCWs positive for HBsAg and/or anti-HBc, 14 (10 %) had positive HBVDNA by PCR. HBV/E (n = 7), HBV/D (n = 3) and co-infection with E and D (n = 4) genotypes were detected. Conclusion: Egyptian HCWs have a significantly high rate of HBV exposure. The detection of HBV/E genotype among Egyptian HCWs suggests prevalent transmission of HBV/E among Egyptian populations.