Health care workers (HCWs) are at high risk of exposure to hepatitis B virus (HBV) and hepatitis C virus (HCV) transmission due to injuries and frequency of exposure. We aimed to assess HBV/HCV seroprevalence among nurses and housekeepers in Beni-Suef University Hospital, and to identify possible risk factors.A cross sectional study was conducted from March to July 2016 using a self-administered questionnaire. A blood sample was withdrawn from each participant and tested for hepatitis B surface antigen (HBsAg) and HCV antibodies. Enzyme-linked immunosorbent assay (ELISA) seropositivity to HCV was confirmed by polymerase chain reaction (PCR).The study involved 175/255 participants with a response rate of 68.5%. Nurses constituted 76% and 24% were housekeepers. Overall prevalence of HCV seropositivity was 4.6%, detected by ELISA. Confirmatory PCR testing revealed positivity in 75% (6/8) of these. Cut injuries were a risk factor for HCV positivity (OR 4.388, 95% CI 0.859 -22.4, P= 0.05). Previous training and use of gloves was a protective factor (OR 0.135, 95% CI .016-1.118, P= 0.03 and OR 0.241, 95% CI 0.055-1.04, P= 0.04 respectively). None of the participants was found to be HBV seropositive. Practices and behaviours posing risk for HCWs included needle stick injury (NSI) and cut injuries. Focus on improving safety training programs to HCWs and provision of infection prevention equipment is needed. In addition regular reporting, follow up and assessment of occupational exposures should be in place.