In this study, the effect of adaptation of B. subtilis to sphalerite and galena with respect to the selective flotation of sphalerite from a sphalerite-galena synthetic mixture has been examined. The changes in the surface charge of the mineral and the bacterium, cell wall components and the profiles of secreted proteins are discussed. The protein profiles of the unadapted and adapted cells are found to differ distinctly, both qualitatively and quantitatively, with impact on the selective flotation of sphalerite and galena. Electrokinetic measurements show shifts in the iso-electric points of not only B. subtilis but also of the chosen minerals after adaptation. Additionally, the zeta potential of B. subtilis is found to become less negative after bacterial adaptation to the chosen sulfide minerals, while it showed an increase in the magnitude of surface negative charge after various enzymatic treatments. The change in the surface morphology and cell wall components such as phosphate, uronic acid and acetylated sugars of the bacterial species during adaptation to sphalerite and galena has been assessed. Selective flotation tests on a synthetic mixture of galena and sphalerite confirm that sphalerite can be preferentially floated from galena in the presence of the insoluble fraction of lysed B. subtilis cells initially adapted to sphalerite, with a high selectivity index.