Microorganisms participate in the soil biogeochemical cycle. Therefore, investigating variations in microbial biomass, composition, and functions can provide a reference for improving soil ecological quality due to the sensitivity of microorganisms to vegetation coverage changes. However, the differences in soil microorganisms between shrubland and meadow have not been investigated in ecologically vulnerable subalpine areas. This study aimed to investigate the biochemical composition and functions of the soil microbial community under two shrublands and a meadow at high altitudes (3,400–3,550 m). Three sites under two shrublands, Rhododendron thymifolium (RHO) and Potentilla fruticosa (POT), and one meadow dominated by Kobresia myosuroides (MEA), were selected on the southern slope of the Qilian Mountains on the northeastern edge of the Qinghai–Tibetan Plateau, China. Soil physicochemical properties, the microbial community composition expressed by the phospholipid fatty acid (PLFA) biomarker, and enzyme activities were analyzed as well as their relationships. The results showed that water holding capacity and the soil carbon, nitrogen, and potassium content in RHO and POT were higher than those in the MEA. Moreover, the soil active carbon, dissolved organic carbon, total nitrogen, and dissolved total nitrogen content in RHO were higher than those in POT. The abundance of total PLFAs, bacteria, and fungi beneath the shrublands was considerably higher than that in the MEA. The PLFA abundance in RHO was significantly higher than that in POT. The fungal-to-bacterial ratio of RHO and POT was significantly higher than that in the MEA. The activities of β-glucosidase, cellobiohydrolase, and leucine aminopeptidase were the highest in RHO among the three vegetation types, followed by POT and MEA. The redundancy analysis indicated that the biochemical composition of the soil microorganisms and enzyme activities were driven by total nitrogen, dissolved organic carbon, water holding capacity, and soil organic carbon. Therefore, shrublands, which have higher biomass, can improve soil moisture status, increase soil carbon and nitrogen content (especially active carbon and active nitrogen), and further increase the abundance of total PLFAs, bacteria, and fungi. The increase of microbial biomass indirectly enhances the activity of relevant soil enzymes. The variations in PLFA abundance and enzyme activities can be attributed to shrub species, especially evergreen shrubs, which create more favorable conditions for soil microorganisms. This study provides a theoretical basis for investigating the soil biogeochemical cycle and a scientific basis for soil management and vegetation restoration in the subalpine regions. Copyright 2022 Fan et al.