Global environmental factors impact soil microbial communities and further affect organic matter decomposition, nutrient cycling and vegetation dynamic. However, little is known about the relative contributions of climate factors, soil properties, vegetation types, land management practices and spatial structure (which serves as a proxy for underlying effects of temperature and precipitation for spatial variation) on soil microbial community composition and biomass at large spatial scales. Here, we compared soil microbial communities using phospholipid fatty acid method across 7 land use types from 23 locations at a regional scale in northeastern China (850 x 50 km). The results showed that soil moisture and land use changes were most closely related to microbial community composition and biomass at the regional scale, while soil total C content and climate effects were weaker but still significant. Factors such as spatial structure, soil texture, nutrient availability and vegetation types were not important. Higher contributions of gram-positive bacteria were found in wetter soils, whereas higher contributions of gram-negative bacteria and fungi were observed in drier soils. The contributions of gram-negative bacteria and fungi were lower in heavily disturbed soils than historically disturbed and undisturbed soils. The lowest microbial biomass appeared in the wettest and driest soils. In conclusion, dominant climate and soil properties were not the most important drivers governing microbial community composition and biomass because of inclusion of irrigated and managed practices, and thus soil moisture and land use appear to be primary determinants of microbial community composition and biomass at the regional scale in northeastern China.