Background Hepatitis B virus (HBV) is a vaccine-preventable infection that can spread in healthcare setting. Data on HBV infections and vaccine in African healthcare workers (HCWs) are limited. We estimated HBV infection prevalence, hepatitis B vaccination status and identified factors associated with vaccination in one Kenyan county. Methods Randomly selected HCWs completed a questionnaire about HBV exposure and self-reported immunization histories, and provided blood for testing of selected HBV biomarkers to assess HBV infection and vaccination status: HBV core antibodies (anti-HBc), HBV surface antigen (HBsAg) and HBV surface antibodies (anti-HBs). Prevalence odds ratios (OR) with 95% confidence intervals (95% CI) were calculated to identify factors associated with vaccination. Results Among 312 HCWs surveyed, median age was 31 years (range: 19–67 years). Of 295 blood samples tested, 13 (4%) were anti-HBc and HBsAg-positive evidencing chronic HBV infection; 139 (47%) had protective anti-HBs levels. Although 249 (80%) HCWs received ≥1 HBV vaccine dose, only 119 (48%) received all three recommended doses. Complete vaccination was more likely among those working in hospitals compared to those working in primary healthcare facilities (OR = 2.5; 95% CI: 1.4–4.3). Conclusion We recommend strengthening county HCW vaccination, and collecting similar data nationally to guide HBV prevention and control.