Microbial electrochemical system employs electroactive microorganisms as catalysts to generate energy and remove pollutants. The formation of electroactive biofilms (EABs) is essential to efficient pollutant removal as it provides the opportunity for microbial interspecies interaction. Bioavailable carbon source serves as the material basis for the energy supply, and its concentration plays a decisive role in EAB formation and pollutant removal. Unfortunately, the mechanisms of how bioavailable carbon source concentration affects microbial interspecies communication and metabolic activity during EAB formation and pollutant degradation have not been thoroughly investigated. Here, phenol was used as the pollutant to evaluate the performance of EABs formed at different acetate loadings (0.2, 0.5, and 1.0 g/L, marked as EAB-0.2 to 1.0). More phenol degrading microorganisms were enriched in EAB-0.2, achieving better phenol removal. Limited bioavailable carbon source forced microorganisms to adjust metabolites, thus facilitating the interspecies interaction. The close linkage between the up-regulated metabolic pathways facilitated the tricarboxylic acid cycle, leading to faster substrate metabolism. We demonstrated that EAB cultivated in a limited organic environment was more active in pollutant removal, which was essential to guide the application of MES in industrial wastewater treatment.