Background: In limited-resource countries, the prevalence of hepatitis B virus (HBV) infection can be measured using data taken from blood donors. This study aimed to determine the HBV infection seroprevalence and its associated factors among blood donors in Sana’a Governorate, Yemen. Methods: A cross-sectional study was conducted on 500 people who donated blood at hospitals located in Sana’a between August and October 2016. The volunteers were aged 18–65 years old and were recruited using convenience sampling. A set of questionnaires was administered through face-to-face interviews. Blood samples from each donor were tested for hepatitis B surface antigens using enzyme-linked immunosorbent assay. Results: The overall seroprevalence of HBV infection in the blood donors was 2.6%. Participants with a history of dental treatment had 7.80 higher odds (95% confidence interval [CI]: 2.26, 26.71; P = 0.002) for HBV infection. Respectively, blood donors who had history of malaria infection or cupping therapy had 7.48 (95% CI: 1.75, 31.82; P = 0.010) and 7.32 (95% CI: 1.72, 30.83; P = 0.010) greater odds of HBV infection. Conclusion: The seroprevalence of HBV infection in Sana’a is lower than in other governorates in Yemen. Stakeholders could focus on a history of dental procedure, malaria infection and cupping treatment when strategising about HBV infection prevention and control among blood donors.