The aim of this study was to determine the effect of substrate concentration (elemental sulfur) on remobilization of heavy metals from mine tailings by indigenous sulfur-oxidizing bacteria. Also, the variation in the binding forms of heavy metals before and after bioleaching was explored. This work showed the laboratory results of bioleaching experiments on Pb-Zn-Cu mine tailings. The results showed that 97.54% Zn, 97.12% Cu, and 44.34% Pb could be removed from mine tailings by the bioleaching experiment after 13 days at 2% w/v substrate concentration. The results also indicated that substrate concentration 2% was found to be best to bacterial activity and metal solubilization of the five substrate concentration tested (0.5%, 1%, 2%, 3%, and 5%) under the chosen experimental conditions. The bioleaching had a significant impact on changes in partitioning of heavy metals.