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Electroactive biofilms (EABs) have aroused wide concern in waste treatment due to their unique capability of extracellular electron transfer with solid materials. The combined effect of different operating conditions on the formation, microbial architecture, composition, and metabolic activity of EABs are still unknown. In this study, the impact of three different factors (anode electrode, substrate concentration and resistance) on the acclimation and performance of EABs was investigated. The results showed that the shortest start-up time of 127.3 h and highest power density of 0.84 W m− 2 were obtained with carbon brush as electrode, low concentration of substrate (1.0 g L− 1) and 1000 Ω external resistance (denoted as N1). The EABs under N1 condition also represented strongest redox capacity, the lowest internal resistance, and close arrangement of bacteria. Moreover, the EABs cultured under different conditions both showed similar results, with direct electron transfer (DET) dominated from EABs to anode. Microbial community compositions indicated that EABs under N1 condition with lowest diversity and highest abundance of electroactive bacteria (46.68%). Higher substrate concentration (3.0 g L− 1) promoted the proliferation of some other bacteria without electroactivity, which was adverse to EABs. The metabolic analysis showed the difference of genes related to electron transfer (Cytochromes C and pili) and biofilm formation (xap) of EABs under different conditions, which further demonstrated the higher electroactivity of EABs under N1. These results provided a comprehensive understanding of the effect of different operating conditions on EABs including biofilm formation and electrochemical activity.