Generally, high bioelectroactivity of anodophilic biofilm favors high power generation of microbial fuel cell (MFC), however, it is not clear whether it can promote denitrification of MFC synchronously. In this study, the impact of anodophilic biofilms bioelectroactivity on denitrification behavior of single-chamber air-cathode MFC (SAMFC) in steady state was studied for the first time. Anodophilic biofilms of various bioelectroactivity were acclimated at conditions of open circuit (OC), Rext of 1000Ω and 20Ω (denoted as SAMFC-OC, SAMFC-1000Ω and SAMFC-20Ω, respectively) and run for 100 days in the presence of nitrate. Electrochemical tests and microbial analysis results showed that the anode of the SAMFC-20Ω delivered higher oxidation and denitrification current response and had a higher abundance of electroactive bacteria, like Geobacter, Pseudomonas and Comamonas, which possessed bidirectional electron transfer function, demonstrating a higher bioelectroactivity of the anodophilic biofilm. Moreover, these electroactive bacteria favored the accumulation of denitrifers, like Thauera and Alicycliphilus, probably by consuming trace oxygen through catalyzing oxygen reduction. The SAMFC-20Ω not only delivered a 61.7% higher power than the SAMFC-1000Ω, but also achieved a stable and high denitrification rate constant (kDN) of 1.9, which was 50% and 40% higher than that of the SAMFC-OC and SAMFC-1000Ω, respectively. It could be concluded that the high bioelectroactivity of the anodophilic biofilms not only favored high power generation of the SAMFC, but also promote the growth of denitrifers at the anodes and strengthened denitrification. This study provided an effective method and important theoretical basis for enhancing power generation and denitrification performance of the SAMFC synchronously.