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Background Hepatitis B (HB) infection is common in Mali. However, there is little information on molecular and biochemical characteristics of HB carriers. Methods A group of 1466 adult volunteers was recruited in the district of Bamako. Confirmed HB carriers were tested for HB viral load by quantitative PCR and HBV was genotyped by sequencing of HBS. Fibrosis and hepatitis activity were measured using the Fibrotest-Actitest. A mutation of TP53 at codon 249 (R249S), specific for exposure to aflatoxin, was detected in cell-free DNA extracted from plasma. Results Overall, 276 subjects were HBsAg-positive (18.8%). Among 152 subjects tested for HBV load, 49 (32.2%) had over 104 copies/mL and 16 (10.5%) had levels below the limit of detection. The E genotype was found in 91.1% of carriers. Fibrotest scores ≥ F2 were observed in 52 subjects (35.4%). Actitest scores ≥ A2 were detected in 15 subjects (10.2%) and were correlated with Fibrotest scores (p = 0.0006). Among 105 subjects tested, 60% had detectable levels of R249S copies (>40 copies/mL plasma). Conclusion Chronic HB carriage in adults in Bamako district is well over epidemic threshold. About 1/3 of carriers have moderate to severe liver fibrosis and 60% have detectable aflatoxin-related TP53 R249S mutation. These results support introduction of anti-HB therapies to reduce the progression towards severe liver disease.