The present study investigates the influence of ferrous iron (as FeSO4) and ferric iron (as Fe-2 (SO4)(3)), and pyrite (FeS2) on the ability of bacterial leaching of a high-grade sulfide Zn-Pb ore. In this regard, shake flask experiments were carried out at 5% (w/v) pulp density of the ore sample (having 40.7% Zn and 12.4% Pb initial metal content) using a consortium of mesophilic iron and sulfur-oxidizing acidophiles. A concentration of 0.04 mol/L of ferric iron in the leaching media was found to be optimum for zinc extraction without affecting growth of the microorganisms. Under this concentration, the dissolution of Zn, Pb, Cd, and As was found to be 57%, 0.2%, 0.03%, and 9.9% in 25 days. Using ferrous iron in the media, 0.16 mol/L of Fe2+ was found to be the optimum concentration for efficient bacterial growth and metal dissolution (54.6% Zn, 0.08% Pb, 0.03% Cd, and 10.2% As) from the sample in 25 days. On the other hand, using pyrite as the source of energy for bacterial growth, an initial 12-day lag period was observed when compared to the effect of ferrous iron in the media. Under the optimum concentration (test with 0.24 mol/L iron in the form of pyrite), the dissolution of Zn, Pb, Cd, and As was found to be 39.8%, 0.1%, 0.03%, and 10% in 25 days. The surface chemistry analysis indicated formation of a sulfur layer over the particle surface that hindered reagent diffusion and affected metal recovery through bioleaching.