[Background] The vast majority of soil microorganisms exist in the form of biofilms. The available studies mainly focus on the impacts of different agronomic measures on the structures of soil bacterial communities, while the mechanisms of different agronomic measures in regulating the formation of multispecies biofilms remain unclear. [Objective] To explore the effects of different agronomic measures on the formation of multispecies biofilms in paddy fields. [Methods] Fresh rice rhizosphere soil samples were collected from the fields with long-term application of lime (SH), organic fertilizer (SY), and no fertilizer (CK). The microbial communities were extracted as the initial inocula to cultivate multispecies biofilms. The in-situ detection techniques such as attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and confocal laser scanning microscopy (CLSM), were combined with crystal violet staining to reveal the effects of changes in soil physiochemical properties and environmental factors (pH and temperature) on the formation of multispecies biofilms. [Results] The soil multispecies biofilms under both agronomic measures had two growth cycles. The optimal growth temperature for the development and maturity of multispecies biofilms was 25 °C. As the pH value increased, the soil multispecies biofilm biomass showed a trend of first increasing and then decreasing. The biofilm-forming ability in the lime application system was significantly higher than that in the organic fertilizer application system, and the multispecies biofilm biomass followed the trend of SH>SY>CK. [Conclusion] Both agronomic measures promote the formation of multispecies biofilms in paddy fields.