Biofilms are widespread in natural and engineering environments and are being increasingly used for pollutant treatment. However, low temperature is one of the major challenges in practice. Here, we developed an alternative strategy to enhance biofilm formation and biofilter startup at 15 °C by supplementing with spermidine, an essential substance for cell growth. A significantly increased biofilm biomass was observed in the treatment group (with spermidine), accompanied by a higher live cell ratio and biofilm matrix excretion as compared with the control (without spermidine). The bacterial communication ability, including quorum sensing and the second messenger system, was found to be enhanced in the treatment group. The bacterial secretion system and quorum sensing pathway were also significantly enriched in differential genes, and most of these genes were upregulated in the treatment group. Robustness and null model analysis revealed that spermidine bolstered microbial community stability and mitigated the dispersal limitation process, which would be conducive to biofilm development. The application of spermidine in biofilters enhanced the biomass accumulation rate and performance. Overall, our results highlighted the importance of the microbial community on biofilm formation regulation and provided a feasible strategy for the enhancement of biofilm formation in low-temperature environments.