A dominant Acidithiobacillus ferrooxidans ssp. was isolated from the supergene copper deposit in Morenci, Arizona, USA. Washed bacterial suspensions (108 MPN per treatment), in pH-neutral buffer, were inoculated onto pyrite cubes for 24 h. Heterogeneous bacterial absorption onto the pyrite removed approximately 90% of the viable bacteria from the inoculum. At T = 0, the bacteria were observed primarily in regions enriched in phosphorus. Over 30 days, the bacterial population on the pyrite cubes increased from 1.3 × 107 to 2.9 × 108 bacteria cm−2. During this growth stage, low levels of thiobacilli (228 ± 167 MPN mL−1) were also recovered from the fluid phase; however, this population decreased to zero within 30 days. Growth on pyrite occurred as micrometre-scale planar microcolonies, a biofilm, coating the mineral surfaces. These microcolonies possessed viable thiobacilli, even after 4 months at ‘circumneutral pH’. Imaging the pyrite cubes using SEM-EDS and scanning force microscopy demonstrated that the thiobacilli grew as iron oxy-hydroxide-cemented cells, leading to the formation of mineralized microcolonies. Removing the iron oxy-hydroxides with oxalic acid did not dislodge the bacteria, demonstrating that the secondary minerals were not responsible for ‘gluing’ the bacteria to the pyrite surface. Removing organic material, i.e. the cells, by an oxygen plasma treatment revealed the presence of corrosion pits the size and shape of bacteria. Because of the inherent geochemical constraints on pyrite oxidation at neutral pH, the colonization of pyrite under circumneutral pH conditions must be facilitated by the development of an acidic nanoenvironment between the bacteria and the pyrite mineral surface.