Soil microbes are critical to the maintenance of forest ecosystem function and stability. Forest diversification, such as monocultures versus mixed forests stands, can strongly influence microbial community patterns and processes, as well as their role in soil ecosystem multifunctionality, such as in subtropical forest ecosystems. However, less is known about these patterns and processes vary with soil depth. Here, we investigated the results of an eight-year forest diversification field experiment comparing the soil ecosystem multifunctionality, bacterial and fungal community assembly, and network patterns in mixed versus monoculture plantations along vertical profiles (0–80 cm depth) in a subtropical region. We found that the introduction of broadleaf trees in coniferous monocultures led to enhanced synergies between multiple functions, thus improving soil multifunctionality. The effects of mixed plantations on the functional potential in top soils were greater than in deep soils, especially for carbon degradation genes (apu, xylA, cex, and glx). Microbial community assembly in the top layer, particularly in mixed plantations, was dominated by stochastic processes, whereas deterministic were more important in the deep layer. Soil microbial network complexity and stability were higher in the top layer of mixed plantations, but in the deep layer was monoculture. Interestingly, the changes in microbial communities and multifunctionality in the top layer were mainly related to variation in nutrients, whereas those in the deep were more influenced by soil moisture. Overall, we reveal positive effects of mixed forest stands on soil microbial characteristics and functionality compared to that of monocultures. Our findings highlighted the importance of enhancing functional diversity through the promotion of tree species diversity, and managers can better develop forest management strategies to promote soil health under global change scenarios.