Background: Health care workers (HCWs) wherever they work are at high risk of trapping infectious blood-borne pathogens, including hepatitis C virus (HCV). Epidemiological studies are needed to focus on HCWs' work profiles which represent the major threat to their health. Objective(s): To determine the prevalence and associated risk factors of HCV seropositivity among HCWs in Aswan University Hospital, Aswan city, Egypt. Methods: A cross-sectional study was carried out during the year 2018 on 206 HCWs selected randomly by proportionate allocation. Data were collected through direct interviews and blood samples were obtained for the detection of HCV antibodies by an enzyme-linked immunosorbent assay (ELISA) test. Results: Out of 206 screened HCWs, 14 of them were positive HCV-Ab giving a prevalence of 6.8%. Cleaning workers had the highest proportion of HCV seropositivity. The significant predictors of HCV-Ab seropositive test were infrequent hand washing after blood or body fluid exposure (OR= 5.12), inconsistent use of personal protective equipment (OR= 4.94), prolonged work duration (OR= 3.93), and having a family history of HCV infection (OR= 3.50). Conclusion: The seroprevalence of HCV among HCWs is alarmingly high. In view of our results, adherence to hand-hygiene, proper use of personal protective equipment and regular screening for HCWs should be strongly emphasized.