The mineralosphere is an important micro habitat in terrestrial ecosystems. How different groups of microorganisms colonise mineral surfaces and whether the level of grassland land-use intensity (LUI) modifies this micro-habitat is not well known, however. We exposed mesh containers filled with pristine soil minerals (illite/goethite) mixed with 13C labelled root litter of Dactylis glomerata and Lolium perenne in grassland soils of the “Schwäbische Alb” (Germany) to characterise the succession of different microbial properties in the mineralosphere. The use of sites within the Biodiversity Exploratories made it possible to select five sites of low LUI and five sites of high LUI. After 1, 2, 7, 12 and 31 months of exposure in the grassland soils, we used physiological, microbiological and isotopic methods to elucidate in situ colonisation patterns, carbon use and levels of extracellular enzyme expression by soil microorganisms associated with mineral surfaces. Microorganisms slowly colonised pristine mineral surfaces and established functionally distinct communities over time. Fungi colonised mineral surfaces to a greater extent than bacteria, reaching 13.2% of control soils compared to 3.2% by bacteria after 31 months. Fungi also reached pristine mineral surfaces earlier than bacteria, probably due to their hyphal growth strategies, and made immediate use of the added complex root litter substrate. This result is evident by the incorporation of up to 74% root litter-derived C into the fungus-specific PLFA (18:2ω6,9) compared to 51% root litter-derived C in the bacteria-specific PLFAs. Both bacteria and fungi associated with minerals remained in an active state (high biomass-specific respiration, high bacterial and fungal growth rates) throughout the experimental period. Grassland LUI and physico-chemical properties of the adjacent soil modified both quantity and quality of substrates available to soil microorganisms in the mineralosphere. Since 13C incorporation into microbial biomass was greater under low LUI than under high LUI, we conclude that microorganisms in low LUI sites had to rely on the added root material, while the carbon signal in microorganisms in the high LUI sites was diluted by alternative sources resulting from transport of dissolved organic carbon into the mineralosphere.