【Objective】Soil minerals, as the main carrier of biofilms, regulate the formation of multi-species biofilms in soil and profoundly affect the types of bacterial interactions within the biofilm.【Method】To further reveal the interaction mechanism between soil active components and multi-species biofilms, this study selected common minerals such as kaolinite, montmorillonite, and goethite in soil, as well as strains extracted from paddy soil, as research objects. Single strains were paired and co-cultured with soil minerals. In situ monitoring was carried out using confocal laser scanning microscopy (CLSM) combined with the crystal violet staining method, to explore the formation of multi-species biofilms and the response of bacterial species interactions within biofilms to soil minerals.【Result】The results show that compared with the pure bacterial system, both kaolinite and montmorillonite treatments significantly inhibited the formation of multi-species biofilms, and the S-1+S-14 combination in the kaolinite treatment system was most inhibited, with a decrease of 42.57% in biofilm biomass. The addition of kaolinite changed the interaction between the S-1+S-2 strain combination from synergistic to neutral and adjusted the S-1+S-8 combination from neutral to antagonistic. However, montmorillonite alleviated the antagonistic interaction between the S-1+S-14 and S-1+S-15 strains, and their interaction relationship shifted to neutrality. The treatment of goethite significantly promoted the formation of multi-species biofilms in the four groups, with the S-1+S-14 combination showing the greatest increase in biofilm biomass (46.45%). Also, the addition of goethite significantly enhanced the synergistic effect of the S-1+S-2 mixed microbial community, causing the interaction between microbial communities in the S-1+S-8 and S-1+S-14 combinations to shift from neutral and antagonistic to synergistic, and adjusting the S-1+S-15 combination from antagonistic to neutral.【Conclusion】This study clarifies the effects of different types of soil minerals on the formation of multi-species biofilms and reveals the potential mechanisms for the transformation of interactions between microbial communities within biofilms. The results of this study can provide theoretical guidance for a deeper understanding of the microbial effects of soil components and soil biological processes, as well as further exploration of soil biological resources.