Biochar is extensively used in the remediation of pollutants because of its diverse physicochemical properties. Biochar application can alter the activity of microbial communities involved in bioremediation. However, the electrochemical behavior of biochar and its potential effect on microbial nitrate reduction remains unknown. Electron transfer between microbial cells and electron donor or acceptor species is often across extracellular polymeric substances (EPS). However, the role of EPS in extracellular electron transfer remains unclear. In this study, we examined the electrochemical behavior of biochar and its effects on microbial nitrate reduction to elucidate the role of EPS in extracellular electron transfer (EET). The biochar prepared by the pyrolysis of Aspen wood chips at 400–600 °C. Electrochemical analysis using cyclic voltammetry, electrochemical impedance spectrum, and chronoamperometry showed that biochars could donate and accept electrons. BC 400, BC 500, and BC 600 had an electron donating capacity of 1.03 mmol e− g−1, 0.9 mmol e− g−1, and 0.8 mmol e− g−1, respectively. Furthermore, biochars prepared at 400 °C significantly enhanced the microbial nitrate reduction process. The phenol Osingle bondH and quinone Cdouble bondO surface functional groups on the biochar probably contributed to the overall electron exchange, and this accelerated the nitrate reduction. Electrochemical analysis revealed that the outer membrane c-type cytochrome and flavin proteins from the biofilm were involved in the electron transfer process, with the EPS acting as a transient media for the microbially-mediated EET. Overall, this study suggested that biochar may be effectively used as an eco-friendly material for the enhancement of microbial denitrification.