BACKGROUND Blood-borne pathogens such as human immunodeficiency virus (HIV), hepatitis B and C (HBV and HCV) viruses and Treponema pallidum remain a major public health problem in sub-Saharan Africa. The purpose of this study was to assess the frequency and clinical implications of HIV, HBV, HCV and Treponema pallidum markers in blood donors in a rural area of Southeast Gabon (Koula-Moutou) from 2012 to 2017. METHODS Hepatitis B surface antigen (HBsAg), anti-HIV, anti-HCV and anti-Treponema pallidum antibodies were screened using rapid diagnostic tests (RDTs). RESULTS Of a total of 5,706 blood donors, 1,054 (18.5%) were seropositive for at least one infectious marker and 59 (5.6%) had serologic evidence of multiple infections. The overall seroprevalence of HIV, HBsAg, HCV, and syphilis was 3.1%; 5.9%; 6.2% and 3.3%, respectively. HIV, syphilis and HCV distributions were associated with neither the sex nor the age of the donors. Only HBsAg seroprevalence was significantly higher in donors of the age group 26-35 years old compared to donors of the age group 36-45 years (OR = 1.43 (95% CI: 1.01-2.04), P = 0.045). There was a significant increase in the frequencies of HIV and syphilis and a regression of HBsAg and HCV among blood donors. CONCLUSIONS This study presents the epidemiology of the main pathogens detected in blood donors in a rural area in Gabon. We found that the overall distribution of transfusion transmitted infectious diseases were lower than those observed in the general population but could be underestimated due to the use of RDTs in the screening process of the blood donations.