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The production of large volumes of waste flotation tailings results in environmental pollution and presents a major ecological and environmental risk. This study investigates bioleaching of waste flotation tailings using Acidithiobacillus ferrooxidans. The experiments were performed with 5.00% solid concentration, pH 2.0 with 100 mL medium for 25 d in the lab. The pH, OPR, metal concentration, dissolved organic matter (DOM) in leachate and extracellular polymeric substances (EPS) were recorded. Bioleaching tailing materials were finally characterized. Results showed that microorganisms, acclimating with mine tailings, effectively accelerated the bioleaching process, achieving maximum Zn and Fe extraction efficiencies of 95.45% and 83.98%, respectively, after 25 days. Compared with raw mine tailings, bioleaching could reduce 96.36% and 95.84% leachable Zn and Pb, and Pb presented a low risk (4.13%), while Zn, Cu, and Cr posed no risk (0.34%, 0.64%, and 0%). Toxicity and environmental risk analysis revealed bioleaching process significantly reduced the environmental risk associated with mine tailings. EPS analysis indicated that the loosely-bound EPS (LB-EPS) and tightly-bound EPS (TB-EPS) fractions contained different organic substances, which played different roles in the bioleaching process. Pearson correlation analysis revealed that EPS was highly correlated with bioleaching behavior (p 0.05), and EPS was the main factor affecting the bioleaching process, promoting bioleaching in the LB-EPS and TB-EPS fractions.