Severe seasonal droughts driven by global climate change significantly alter the cycling of carbon and nutrients in forest ecosystems, while the investigation into the impacts of floor mass and plant roots on soil microbial biomass within the context of recurrent seasonal droughts is still rare. To investigate the environmental determinants governing soil microbial biomass with the escalating severity of seasonal droughts, we conducted a study in a montane subtropical moist evergreen broad-leaved forest in southwestern China from June 2019 to May 2023. The study results revealed that soil microbial biomass, as well as soil moisture, floor mass, and plant roots, showed an apparent single-hump modal within one year. In the comparative analysis of the soil microbial biomass fluctuation amplitudes across control and watered plots, a discernible disparity was observed, indicating significant differences in microbial biomass dynamics between the respective experimental conditions. The pooled data revealed a statistically significant influence of seasonal drought, floor mass, plant roots, and their reciprocal interactions on the soil microbial biomass, highlighting these factors as pivotal determinants of microbial community dynamics. This study elucidates the interactive regulatory mechanisms by which seasonal drought, floor mass, and plant roots collectively modulate soil microbial biomass within tropical and subtropical forests, offering insights into the complex ecological processes governing microbial community dynamics. This interactive regulation might influence the trajectory of plant species and soil microbial communities, facilitating their adaptive development and evolutionary responses.