Background Soil microorganisms drive subsurface ecological processes and are shaped by soil properties, which in turn influence biogeochemical cycling. Although the link between biodiversity and soil multifunctionality (SMF) has received widespread attention, the relative roles of soil properties and microbial community structure in regulating SMF remain unclear. Methods Here, we investigated SMF and microbial communities in the humus layer and across the 0–80 cm depth of natural soil profiles in a subalpine grassland using high-throughput sequencing. Results Our findings revealed that SMF, microbial diversity and network complexity decreased significantly with soil depth. Microbial community structure was primarily determined by pH and soil water content (SWC). Soil properties were the primary drivers of SMF, predicting 47.24%–63.75% of its variance. Microbial diversity was a stronger predictor of SMF than network complexity, explaining 26.09–44.56% of its variation. Bacterial diversity was significantly positively correlated with soil nutrient, carbon and nitrogen multifunctionality, while fungal diversity was not significantly correlated with them. This finding provides critical data support for elucidating the relationship between biodiversity and ecosystem functioning.