Biofilms have been extensively studied in aquatic and clinical environments. However, the complexity of edaphic microenvironment hinders the advances toward understanding the environmental functionalities and ecological roles of soil biofilms. In this work, artificial soil was employed to investigate the soil biofilm formation and corresponding impacts on community structure and microbial activities. Our results showed that extracellular polymeric substances (EPS) production was significantly enhanced and micro-meter sized cell aggregates formed with high glucose amendment. Biofilm development exhibited significant effects on the soil microbial processes. 16S rRNA gene sequencing demonstrated the soils with biofilms and free-living cells shared similar microbial communities. But the Shannon diversity and evenness indices of communities with soil biofilms were significantly enhanced by 18.2% and 17.1%. The soil with biofilms also revealed a rapid response to nutrient provision and robust microbial activity, which consumed 65.4% more oxygen in the topsoil (0–1.5 mm). Kinetic respiration analysis showed that the enhanced metabolic activity was attributed to 23-times more active microbes in soil biofilms. In summary, this study revealed that soil biofilms can sustain a diverse and robust community to drive soil biogeochemical processes.