Acidithiobacillus ferrooxidans at 30 °C and Sulfobacillus thermosulfidooxidans at 47 °C were selected from a preliminary screening of various acidophiles for their ferric iron reduction capacities during anaerobic, autotrophic growth on sulfur. The selected cultures were used with a polymetallic sulfide ore under anoxic conditions to demonstrate enhanced solubilization of iron during leaching in shaken flasks and enhanced removal of iron from laboratory ore-leaching columns, compared to leaching with continuous aeration. Ore-associated, ferric iron-rich precipitates, which were formed under previously oxidizing conditions, were a potential influence on extraction of target metals and percolation through ore columns and were available as the source of ferric iron for anaerobic sulfur oxidation. Over twice as much iron was removed by moderate thermophiles when anoxic phases were introduced during the leaching. Enhanced removal of iron and some improvement in extraction of base metals from ore fragments were also demonstrated with a selected “Sulfolobus”-like strain during growth and leaching with alternating periods of aeration and anoxic conditions at 70 °C.