This study evaluated the effects of temporary external voltage on the performance of two-chambered microbial fuel cells (MFC) that use nitrate wastewater as a substrate. Results indicate that the external voltage affected the performance of the MFC during their operation, and this effect remained even after the voltage was removed. The degradation efficiency of the chemical oxygen demand increased in the MFC under external voltages of 0.5, 0.8, and 1.1 V, and the optimal applied voltage was 1.1 V. Compared with the control group without external voltages, the MFC under a voltage of 1.1 V achieved higher current densities and efficiency of nitrate removal during their operation. The MFC with an applied voltage of 1.1 V also achieved the highest maximum power density of 2,035.08 mW/m3. The applied voltages of 0.5 and 0.8 V exerted a positive effect on the performance of the MFC. High-throughput sequencing was used to explore the anode and cathode biofilms. Results showed that the influence was highly associated with microbial community in bio-anode. The predominant functional family changed from Methanotrichaceae during start-up to Flavobacteriaceae in a steady phase.