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Pyrite oxidation by mixed mesophilic acidophiles was conducted under conditions of controlled and non-controlled redox potential to investigate the role of sessile microbes in pyrite oxidation. Microbes attached on pyrite surfaces by extracellular polymeric substances (EPS), and their high coverage rate was characterized by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). The dissolution of pyrite was negligible if the redox potential was controlled below 650 mV (near the rest potential of pyrite), even though the bacteria were highly active and a high coverage rate was observed on pyrite surfaces. However, with un-controlled redox potential the rate of pyrite oxidation increased greatly with an increasing redox potential. This study demonstrates that sessile microbes play a limited role in pyrite oxidation at a redox potential below 650 mV, and highlight the importance of solution redox potential for pyrite oxidation. This has implications for acid mine drainage control and pyrite oxidation control in biometallurgy practice.