Objectives: this study aimed at determinination of HBV viral markers, vaccination status of physicians and nurses acting in high risk departments and to estimate anti- HBs titer in vaccinated persons. Subjects and methods: A cross sectional study was done for doctors and nurses in high-risk departments in a district hospital in Menoufiya Governorate selected by simple random sampling. One hundred health care workers (HCWs) voluntarily participated in the study. A structured questionnaire was used to collect demographic data, HBV vaccination history and occupational risk factors. ELISA was used to test sera for HBsAg, Anti-HBs and total anti-HBc. Results: among the 100 participants, none of them tested positive for HBsAg, 49% had Anti-HBs, and 26% had Anti-HBc. Only 29% of the HCWs were immune after vaccination although 34% reported complete three doses HBV vaccine. Forty five of HCWs (45%) never vaccinated and were susceptible to HBV infection. The prevalence of life time exposure to HBV was 26%. All who reported complete vaccination had HBsAb, pyears (149.48± 43.67 vs. 97.47±29.25), in those who are free from chronic diseases (144.77± 45.70 vs. 102.08±31.01), and among those who had last dose of vaccine exposure factor. There is a need to vaccinate all HCWs as a matter of policy.