Soil is increasingly under environmental pressures that alter its capacity to fulfil essential ecosystem services. To maintain these crucial soil functions, it is important to know how soil microorganisms respond to disturbance or environmental change. Biofilm production is a common trait among microorganisms in a wide range of environmental, engineered, and host-associated ecosystems. However, as for soil systems, the complexity of edaphic microenvironment hinders the advances toward understanding soil biofilm formation processes, structure and ecological functions. In this work, artificial soil was employed to investigate the soil biofilm formation and corresponding ecological impacts under well-defined conditions. []Our results showed that soil biofilm formed under high-nutrient condition. The soil with biofilm exhibited a rapid response to nutrient provision and robust microbial activity, which depleted oxygen in the topsoil. Kinetic respiration analysis revealed that soil biofilm sustained 40-times more active microbes than that without biofilm, while the specific growth rates were comparable. 16S rDNA sequencing demonstrated similar microbial communities under different nutrient conditions, while the one with biofilm consisted of more diversified populations and enriched Bacilli. Our finding provides novel insights about the environmental prerequisite for soil biofilm formation which sustain a diverse and robust community to impact soil biogeochemical processes.