Health care personnel are at high risk for hepatitis B virus (HBV) transmission. The aim of the present study was to investigate hepatitis B (HB) knowledge and vaccination acceptance among health care personnel in southwestern Greece, using the Hepatitis B Vaccine Knowledge and Acceptance Questionnaire. One hundred eighty-three employees participated (71 males). Occupation (p < .001), higher education (p < .001), and vaccination (p = .007) were significantly related to HB knowledge. The rate of HBV vaccination coverage was 70.9%. Participants considering themselves at high risk for HBV infection did not report significantly increased vaccination rates. In the multivariate analysis, university personnel (p = .002), occupational category (p < .001), and HB knowledge (p = .049) were significantly associated with vaccination. In conclusion, 29% of hospital personnel were not vaccinated mainly due to negligence. Occupation, education, and knowledge about HB seem to be associated with HBV vaccination.