Abstract Background and Objectives Hepatitis B virus (HBV) is the virus transmissible by transfusion in Cameroon with highest prevalence. The benefit of pre‐donation screening (PDS) versus post‐donation testing (PDT) remains to be confirmed because of controversial results produced by the use of different methodologies. This study aims to compare the performance and blood donor acceptability with PDT strategy in the reduction of HBV risk. Materials and Methods A quasi‐experimental study was conducted with two groups of participants. The consenting subjects chose prospectively and randomly either the PDS or the current PDT strategy. The donors included in the PDS group were screened for hepatitis B surface antigen (HBsAg) by using a World Health Organization (WHO)‐prequalified HBsAg assay. The PDT strategy consisted of the routine double HBsAg testing with a combination of a rapid diagnostic test (RDT) and enzyme immuno assay (EIA). HBV confirmatory strategy combined testing for DNA detection and HBsAg neutralization assay. A questionnaire was designed to assess donor and staff satisfaction. Results A total of 2528 blood donors were included in the study, of whom 1146 and 1382 went through PDS and PDT screening, respectively. The sensitivity of PDS and PDT was 100% and 96.4% (95% confidence interval [CI]: 93.1–100), respectively, and the specificity was 99.9% (95% CI: 99.3–100) for the two screening strategies. The overall acceptability score of the donor in the PDS group was 8.16 ± 1.61 versus 8.00 ± 1.55 in the PDT group (odds ratio [OR] 95% CI: 0.16 (0.04–0.28); p = 0.012). Conclusion This study supports the benefits of safety and acceptability of the donor and also possible acceptable cost/benefit of the screening measure in the case of limited resources.