Soil microbial communities play an important role in driving a variety of ecosystem functions and ecological processes and are the primary driving force in maintaining the biogeochemical cycle. It has been observed that soil microbial diversity decreases with land use intensification and climate change in the global background. It is essential to investigate whether the reduction in soil microbial diversity can affect soil multifunctionality. Thus, in this study, the dilution-to-extinction method was used to construct the gradient of soil microbial diversity, combined with high-throughput sequencing to explore the impact of the reduction in bacterial, fungal, and protist diversity on soil multifunctionality. The results showed that the soil microbial alpha diversity (richness and Shannon index) was significantly lower than that of the original soil. Principal coordinate analysis (PCoA) showed that the microbial community structure of original soil was significantly different from that of diluted soil, and the response of bacterial and fungal communities to diluted soil was higher than that of protists. The regression model showed that there was a significant negative linear relationship between the average response value of soil multi-function and the index of microbial diversity, indicating that the change in soil microbial community was the key factor in regulating soil multifunctionality. The regression model showed that there was a significant negative linear relationship between soil multifunctionality and microbial diversity, indicating that the change in soil microbial community was the key factor to regulate soil multi-kinetic energy. Through the aggregated boosted tree analysis (ABT) and regression model, we found that some specific microbial groups, such as the Solacocozyma and Holtermaniella of fungi and Rudaea of bacteria, could significantly promote the change in soil multifunctionality, which showed that key microbial taxa play an indicative role in biological processes. Furthermore, the structural equation model revealed that bacteria could affect soil multifunctionality through the interaction between microbiomes, which was the key biological factor driving the change in soil multifunctionality. This study provided experimental evidence for the impact of soil microbial diversity on soil multifunctionality, and promoted the notion that maintaining a certain diversity of soil microbial community in a single agricultural ecosystem, especially the diversity of key microbial taxa, is of great significance to the sustainable development of ecosystem function in the future.