Microbial fuel cells (MFCs) is promising to combat environmental pollution by converting organic waste to electricity. One critical problem for practical application of MFCs treating wastewater is sluggish oxygen reduction reaction (ORR) on cathode. This study focused on developing novel metal-free cost-effective cathodic catalysts to enhance power generation of MFCs. Specifically, carbon powder (Vulcan XC-72R) was modified with acid treatment and pyrazinamide (as nitrogen precursor), and subsequently pyrolyzed at different temperatures. For CN-X (X = 700-1000 °C) materials, chemical compositions (the doping contents of nitrogen species, oxygen-containing groups, and sulfur-containing groups) were altered with pyrolysis temperature. Linear sweep voltammetry showed that CN-800 exhibited the highest ORR activity, with an onset potential of 0.215 V and a half-wave potential of -0.096 V (vs. Ag/AgCl). Electrochemical measurements clearly presented an enhancement of ORR activity by treating carbon powder with sulfuric acid and nitrogen doping, which was well correlated with voltage output in single chamber MFCs (SCMFCs). On the other hand, for the nitrogen-doped cathode catalysts, the best performance in SCMFCs was directly related with the amount of pyridinic nitrogen species and total nitrogen amount. The MFC operated with CN-800 exhibited a maximum power density of 371 ± 3 mW/m2 with the chemical oxygen demand (COD) removal of 77.2 ± 1.5% and coulombic efficiency (CE) of 8.6 ± 0.3%. Furthermore, the MFC with CN-800 exhibited an excellent stability over longer than 580 h of operation with 1.5% voltage reduction. CN-800 possessed comparable COD removal efficiency to conventional costly Pt/C, and exhibited distinct cost-effectiveness for MFC practical applications in wastewater treatment.