Conditioning films and biofilms forming on surfaces of solid materials exposed to aqueous media play a key role in in the interaction between the geo- and biospheres. In this study, time-of-flight secondary ion mass spectrometry and scanning electron microscopy were used to investigate the time scale, mode of formation, and chemistry of conditioning films and biofilms that formed on Si substrates exposed to aquifer water in the subsurface Äspö Hard Rock Laboratory, SE-Sweden. The detection of fragment ions of amino acids, carbohydrates, and carboxylic acids revealed that different types of organic compounds had adhered to the Si surface already after 10 min of exposure to the aquifer fluids, whereas the attachment of microbial cells was first observed after 1000 min. The organic compounds first formed isolated μm-sized accumulations and subsequently started to distribute on the wafer surface more homogenously. Simultaneously further microorganisms attached to the surface and formed biofilm-like cell accumulations after 3 months of exposure to aquifer water.