Background: Infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is a major public health problem worldwide. The aim of this study was to investigate the epidemiological and diagnostic aspects of HBV and/or HCV infection among healthcare professionals at the CNHU-HKM in Cotonou. Methods: This was a descriptive and analytical cross-sectional study. Data collection was prospective and lasted 2 months, from July 2022 to September 2022 at the university occupational health clinic, then at the university hepato-gastroenterology clinic of the CNHU-HKM in Cotonou. Results: A total of 806 people were included, with a mean age of 43.1 ± 1.9 years and a sex ratio of 1.1. Nurses were in the majority (259; 32.1%), followed by orderlies (185; 23%) and administrative staff (122; 15.1%). HBsAg was positive in 51 subjects, for a prevalence of 6.3%, and anti-HCV antibody in 6 subjects, for a prevalence of 0.7%. Of these positive cases, 23 returned for further investigations, 78.2% of them asymptomatic. Among HBsAg carriers, 13 were chronically HBeAg-negative (59.1%). None of the subjects had cirrhosis or hepatocarcinoma. There were no factors significantly associated with the presence of HBsAg in healthcare professionals. Conclusion: This study shows that hepatitis B and C are common among all health professionals at this hospital, and are usually asymptomatic. Systematic screening (at the time of hiring) is therefore desirable, in order to ensure adequate care and contribute to the eradication of these scourges in Benin.