Land use and management practices have a significant impact on soil microbial populations and community composition, thereby influencing ecosystem processes. Soil microbial and biochemical indicators serve as highly sensitive tools for assessing the effects of land use systems. In this study, we investigated soils under ten different land use systems in the Central Himalayas, India, including natural forests dominated by oak (Quercus incana), deodar (Cedrus deodara), or pine (Pinus roxburghii) trees, orchards dominated by apple trees, and crop-based systems in uplands and valleys. We examined phospholipid ester-linked fatty acid (PLFA) profiles, soil enzymes, Ribosomal Intergenic Spacer Analysis (RISA) profiles, microbial biomass carbon and organic carbon as effected by land use. The results revealed that forest soils had significantly higher levels of soil enzymes compared to agricultural soils. Microbial biomass carbon and organic carbon showed a close relationship with the PLFA profiles across different land uses. Gram-positive bacteria (15:0 iso, 16:1 iso G, 16:1 iso H), gram-negative bacteria (10:0 2OH, 12:0 3OH, 17:0 cyc, 19:0cyc 8c, 18:1ω7c11), fungi (18:1ω9c), and arbuscular mycorrhizal fungi (AMF) (16:1) exhibited higher relative abundance in forest systems, whereas gram-positive PLFA markers (C15:0 anteiso, C17:0 iso, C17:0 anteiso) were more prominent in agro-ecosystems. Principal component analysis (PCA) of the PLFA profiles demonstrated that the microbial communities in deodar forest soils were compositionally distinct from other forest soils, while the cultivated soils were grouped together and exhibited higher similarity, except for the organic farming soil. The correlation between PLFA profiles and soil enzymes, microbial biomass carbon, and organic carbon provides insights into the impact of different land use and management practices on soil microbial health and, consequently, soil health.