The aim of this study was to investigate interspecies interaction by quantifying pyrite dissolution and to visualize the colonization of metal sulfides in pure and mixed cultures of leaching bacteria. Strains, such as chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 23270 (type strain) and chemoorganoheterotrophic Acidiphilium cryptum JF-5, were used. Sessile, pyrite-detached and planktonic cells were visualized by epifluorescence microscopy using DAPI staining and FISH. Additionally, atomic force microscopy was used for investigations on cell morphology, bacterial distribution on pyrite and mineral surface topography. In pure At. ferrooxidans cultures and in mixed cultures with Ap. cryptum JF-5, it could be shown that the bacterial morphology of sessile cells differed significantly from those of planktonic cells by decreased cell sizes and enhanced production of extracellular polymeric substances in case of sessile cells. Interspecies interaction in mixed cultures resulted in increased pyrite leaching and production of extracellular polymeric substances and consequently, enhanced biofilm formation.