Cost-effective cathode catalysts are critical to the development of microbial fuel cell (MFC) technology. Herein, a synthesis route is presented to improve the nitrogen content and nitrogen functionality in the nitrogen-doped activated carbon (AC) as a low cost and efficient catalyst for oxygen reduction reaction (ORR). It was demonstrated that key factors for successful nitrogen doping were the proper pretreatment with acidic and alkaline solutions consecutively and the use of a solid-state nitrogen precursor. The AC pretreated with both acidic and alkaline solutions resulted in a nitrogen content of 8.65% (atom %) (in which 5.56% is pyridinic-N) on its surface, and exhibited an outstanding electrocatalytic activity for ORR in both electrochemical and MFC tests. A good agreement between pyridinic-N content and ORR activity was observed, indicating that pyridinic-N is the most active site for ORR in the nitrogen-doped AC. The pretreated nitrogen-doped AC catalysts resulted in a higher maximum power density than the untreated AC and the commercial Pt/C (10% Pt) catalysts. The exceptional performance associated with the advantages, such as simple and convenient preparation procedure, easily obtained raw materials, and low cost, makes the pretreated nitrogen-doped AC promising for the ongoing effort to scale up MFCs.