Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus associated with coronavirus disease (COVID-19). At the time of the study, little data on the level of exposure of the population in Koutiala district in Mali to SARS-CoV-2 was available. Although blood donors are not representative of the general population, a COVID-19 seroprevalence estimate in this population was intended to assess the extent of community transmission, serve as a health alert system, and help guide the public health response. We measured seroprevalence of anti-SARS-CoV-2 antibodies using NG-Biotech SARS-Cov-2 RDT and ECLIA test between January and June 2020. This is a cross-sectional study of volunteer blood donors aged 18 to 60 years, independent of any previous COVID-19 disease. A stratified analysis of seroprevalence by month of sample collection and a comparison of the results of the NG-Biotech SARS-Cov-2 RDT with those of the ECLIA test was performed. The overall prevalence of antibodies to SARS-Cov-2 virus assessed by the NG-Biotech SARS-Cov-2 RDT was 24.6% (95% CI 21.8–27.4) and by the ECLIA test was 70.2 (95% CI 64.9–75.5). Both estimates remained relatively stable over the study period. We observed SARS-CoV-2 exposure much higher than indicated by case-based surveillance. The national surveillance system, as it was, was not able to detect variations in incidence, and as such, we do not recommend it as an alert system. However, the discrepancy between the results of the rapid test and the ECLIA test shows that further research is required to assess the validity of these test before a more solid conclusion can be drawn it their use in surveillance.