Acid Mine drainage (AMD) is a significant environmental problem. Bacteria play a significant role in causing AMD, since they play a large role in catalyzing the oxidation of pyrite (a root cause of AMD) and the concomitant development of acidic conditions. The chemolithoautotrophic species, Acidithiobacillus ferrooxidans, and the heterotrophic species, Acidiphilium acidophilum were investigated on the pyrite surface with atomic force microscopy (AFM) and aqueous batch reaction rate measurements. AFM was used to investigate the growth of this bacterial species on the pyrite surface at a pH close to 2, with and without an adsorbed organic layer of phosphocholine lipid. Specifically, 1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine lipid (23:2 Diyne PC), a phosphocholine lipid, was used, which based on earlier work showed a strong inhibitory effect on both the abiotic and biotic oxidation rate of pyrite. AFM showed that under the experimental conditions used in this study, the lipid formed a bilayer-structure on the mineral surface, which was stable for long periods (>25 days) of time in the presence of the bacteria. In- situ AFM also showed that the adsorption of the phosphocholine lipid resulted in a decrease in the amount of surface-bound bacteria (relative to the aqueous phase), due to the strong interaction of the phosphate functional group of the lipid and the pyrite surface.