Forest land-use changes have long been suggested to profoundly affect soil microbial communities. However, how forest type conversion influences soil microbial properties remains unclear in Tibetan boreal forests. The aim of this study was to explore variations of soil microbial profiles in the surface organic layer and subsurface mineral soil among three contrasting forests (natural coniferous forest, NF; secondary birch forest, SF and spruce plantation, PT). Soil microbial biomass, activity and community structure of the two layers were investigated by chloroform fumigation, substrate respiration and phospholipid fatty acid analysis (PLFA), respectively. In the organic layer, both NF and SF exhibited higher soil nutrient levels (carbon, nitrogen and phosphorus), microbial biomass carbon and nitrogen, microbial respiration, PLFA contents as compared to PT. However, the measured parameters in the mineral soils often did not differ following forest type conversion. Irrespective of forest types, the microbial indexes generally were greater in the organic layer than in the mineral soil. PLFAs biomarkers were significantly correlated with soil substrate pools. Taken together, forest land-use change remarkably altered microbial community in the organic layer but often did not affect them in the mineral soil. The microbial responses to forest land-use change depend on soil layer, with organic horizons being more sensitive to forest conversion.