Health care workers (HCWs) represent a high-risk population for hepatitis infection. The study aimed to assess the level of knowledge of nurses regarding hepatitis B virus (HBV) infection. A cross-sectional study was conducted from September 2020 to November 2020. A total of 300 nurses were included in the study. Data were collected using a self-administered structured questionnaire, and analyzed by using SPSS (22). The results revealed that the mean knowledge score among nurses was 62.55% of the information tested in our questionnaire. Most of the correct answers were in the nature section (78.4%), while answers were poor on vaccination (46.9%). About 118 (40%) of the nurses aged between 20-30 years, 157 (52%) were females. Two hundred fifty (83%) respondents reported taking the HBV vaccine, and 96.1% reported having titers >10U/I. Based on our findings, nursing knowledge about HBV in prevention and vaccination was unsatisfactory compared with the nature, treatment and transmission. Continuous training programs toward viral infection are highly recommended.