Overall performances of Single Chamber Microbial Fuel Cells (SCMFCs) with air-cathode are extremely improved by optimizing a cathodic catalyst not containing metals. To reach this goal, the main target is based on design nitrogen doped carbon-based nanofibers (N-CNFs) as catalyst layer at the cathode. The fine-tuning of the electrochemical properties of CNFs plays a key role to grant the direct oxygen reduction reaction (ORR). A pyrolysis treatment, conducted under inert atmosphere at 900 degrees C, is carried out to obtain a self-induced nitrogen doping in the carbon nanofibers during carbonization step, ensuring a proper value of electrical conductivity. The resulting N-CNFs, thanks to their content of nitrogen defects together with their high surface area, result to be a promising catalyst layer for the ORR. Physical, chemical and morphological properties of nanofibers are investigated, demonstrating the presence of N-doped graphitic sheets in the nanofiber mats. In particular, the key role of graphitic and pyridinic nitrogen related to the improvement of electrochemical behaviour of N-CNFs, is explained. All these features enhance the overall performance of SCMFCs. Indeed, SCMFCs with N-CNFs catalyst cathode reach a maximum power output comparable to that obtained by Pt based cathodes. The impressive result is strictly due to the important features of N-CNFs. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.