Bioleaching treatment was used to decontaminate pyrite-bearing waste rocks, mitigating the risk of acid mine drainage (AMD) generation at source. The bioleaching results showed that nearly 82% of Fe and S could be removed from pyrite-bearing waste within 40 d in the presence of Acidithiobacillus ferrooxidans (A. ferrooxidans). Kinetics study showed that the removal of pyrite from waste ore was mainly in the control of the chemical reaction and internal diffusion. The results of X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) indicated that the formed biogenic jarosite could passivate the residual pyrite under the mediation of A. ferrooxidans. Secondary bioleaching experiments on the residual samples revealed no noticeable decrease in solution pH and only 8 and 160 mg/L of iron dissolved from the two residual mine waste samples, respectively, indicating that the acid and iron release capacity of the mine waste samples was weakened.