Leaching bacteria attach to their substrates, i.e. mineral sulfides, and form monolayered biofilms. In this study the biofilm formation of Acidithiobacillus ferrooxidans A2 on pyrite was examined using atomic force and epifluorescence microscopy (AFM and EFM, respectively). A novel system by JPK instruments, the BioMaterial WorkstationTM, allows the investigation of the same location on an opaque sample with AFM and EFM. Until recently this was only possible for translucent samples. Sessile bacteria on pyrite coupons were stained with 4’,6-diamidino-2- phenylindol (DAPI) and visualized by EFM as well as AFM. The best imaging conditions for AFM were assessed. Scans of bacteria attached to pyrite were performed in contact mode in air as well as in tapping mode in fluid. Imaging in fluid was more challenging than imaging in air as bacteria tend to detach from their substratum. To avoid the dislocation of microorganisms by the AFM probe the sample was dried in air for 1 h prior to scanning in fluid. Scanning in air was performed with the whole range of cantilever spring constants tested (k = 0.03 N/m to k = 0.65 N/m) while, for scanning in fluid, best results were achieved using stiffer cantilevers (k = 0.65 N/m).