The purpose of this retrospective study, which examined data collected from January 2007 through September 2010, was to evaluate the seroprevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) among 2946 new blood donors at the Nianankoro Fomba Hospital (NFH). The overall seroprevalence of HIV was 0.88%, of HBV 5.3%, and of HCV 0.55%. Donors infected with HIV and HBV were younger than healthy donors, while those infected with HCV were older. These findings at the NFH show that HIV, HBV, and HCV remain major public health problems in Mali.