Vegetation degradation caused by intense human disturbances poses a significant challenge to the preservation and improvement of ecosystem functions and services in the karst region of southwest China. Soil microorganisms are major regulators of ecosystem multifunctionality (EMF). Currently, there is a dearth of knowledge regarding the effects of vegetation degradation on soil microbial communities and their corresponding multiple ecosystem functions in karst regions. In this study, we selected the vegetation degradation sequences of second natural forest (NF), agroforestry (AS) and cropland (CL) to investigate the diversity of bacterial, fungal and protistan communities, and their hierarchical co-occurrence network, and EMF to explore the relationships between them. Compared to the NF, the carbon cycling index, nitrogen cycling index, soil water regulation power, and the EMF were significantly decreased by 8.2%–50.6%, 48.7%–86.8%, 19.8%–24.5%, and 31.4%–69.5% in the AS and CL, respectively. The development of EMF can be explained by the fungal, protistan and microbial hierarchical β-diversity, as well as the complexity (e.g. degree) of microbial hierarchical interactions during the process of vegetation degradation. Notably, correlations between the abundances of sensitive amplicon sequence variants (sASVs) for different karst vegetation types and EMF varied in distinct network modules, being positive in module 1 and negative in module 2. Moreover, the relative abundance of keystone taxa in fungal and protistan communities provided greater contributions to EMF than the bacterial communities. Additionally, random forest modeling showed that carbon and nitrogen sources, and soil water content, and trace elements (e.g. exchangeable magnesium, iron, manganese, and zinc) were identified as key driving factors of the EMF. Collectively, our findings demonstrate that vegetation degradation obviously alters soil microbial diversities and hierarchical interactions, emphasizing their key role in maintaining ecosystem functions and health in karst regions.