Background: Hepatitis B infection is a potentially severe liver infection caused by the hepatitis B virus and is a significant global health issue. The likelihood of health care professionals contracting hepatitis B is high. Vaccination is the most effective and feasible method for avoiding hepatitis B virus infection. Hepatitis B virus (HBV) prevention requires adequate knowledge, attitudes, and practices regarding hepatitis B infection and immunization. Aim of work: To study the level of knowledge, attitudes, and practice towards HBV infection among health care workers in Suez Canal University Hospital. Materials and methods: A descriptive cross-sectional study was conducted on 305 health care employees at Suez Canal University Hospital, including physicians and nurses. A self-administered questionnaire with sociodemographic, knowledge, attitude, and practice-related questions was established. Results: Among the 305 HCWs of the study, 55.5% have adequate understanding of HBV transmission, even though 80% of health care workers (HCWs) were vaccinated against HBV, only 7.5% had immunity test after immunization. About 50% of physicians and 38.7% of nurses sustained 2 to 5 needle stick / sharp injury incidents during their working careers. Nearly, 16.2% of physicians and 19.0% of nurses reported using post-exposure prophylaxis, including hepatitis B vaccination. Conclusion: The majority of HCWs at Suez Canal University Hospital have proper knowledge, attitudes, and practices regarding HBV transmission and immunization. The health care authorities should provide training courses on hepatitis B infection and vaccination for HCWs to reduce the risks of acquiring hepatitis B virus infection.