Soil microbial communities are crucial in enabling ecosystem multifunctionality (EMF). However, the mechanisms by which biotic and abiotic factors regulate EMF in alpine grasslands, and the regulatory effects of soil microorganisms on ecosystem function under the background of altitude at large regional scales, remain unclear. We established a 1500-km sampling area along the altitude gradient in alpine grassland on the Tibetan Plateau. With increasing altitude, bacterial and fungal community diversity increased, and microbial network complexity and EMF showed unimodal variation patterns. Soil bacterial communities were more sensitive to environmental changes than fungal communities. Microbial network complexity was a better predictor of ecosystem functioning than the microbial community composition when both mono- and multifunctional drivers were considered, and annual mean temperature and soil organic carbon were the main environmental drivers. Our study demonstrates the importance of symbiotic networks as key components of microbial biomes for climate change buffering effects in alpine grasslands.