We aimed to determine hepatitis B virus (HBV) prevalence and risk factors for health care workers (HCWs) to contract HBV infection in a referral hospital in the Democratic Republic of Congo. From October 2015 to August 2016, we recruited 97 HCWs (55 males and 42 females, aged 41.2 ± 10 yr) from Kisantu St. Luke's Hospital, a Congolese referral hospital located in the province of Kongo Central. Serum samples were assayed for HBV markers using ELISA. A questionnaire was used to record the HCWs' demographics, medical histories, and risk factors. The overall prevalence of exposure to HBV infection [HBsAg+, and/or hepatitis B core antibody (anti-HBc)+] was 56.7% (55/97). HBsAg positivity was found in 18.6% (18/97) of the HCWs whereas 29.9% (29/97) were anti-HBc positive. Approximately 8.2% (8/97) of the HCWs tested positive for both HBsAg and anti-HBc. Being a physician [odds ratio (OR)=2.8 (95% CI: 1.34-12.23)], a laboratory technician [OR=3.35 (95% CI: 1.35-5.21)], and having multiple sex partners [OR=3.05 (95% CI: 1.13-9.09)] were found to be factors associated with HBV infection. Exposure to HBV is common among HCWs at Kisantu St. Luke's Hospital. Isolated HBsAg was also prevalent among them. There is a high risk that HBV could be spread to others. Therefore, there is an urgent need for HBV screening, treatment, and vaccination policies.