Developing high-performance cathodic catalysts and exploiting new techniques to investigate the nanoscale process of the oxygen reduction reaction (ORR) are of significant importance for the wide application of microbial fuel cells (MFCs). Here, novel Fe-based clusters embedded in nitrogen doped activated carbon (Fe-clusters/NAC) catalysts were developed via a simple ball milling method. Abiotic electrochemical tests demonstrated that these Fe-clusters/NAC catalysts exhibited an enhanced ORR performance due to the formation of Fe-based clusters, and the exposure of abundant active sites including quaternary N, Fe–N and Fe-based clusters. More importantly, the Fe-clusters/NAC catalysts exhibited an even better ORR performance than Pt due to the existence of more active sites than Pt, which was confirmed by using a scanning electrochemical microscope. Acid etching treatment and density functional theory calculations further verified the superior ORR activity of Fe-based clusters. The power density of the Fe-clusters/NAC catalyst-based MFCs reached 2387 mW m⁻², which is the highest value among the reported Fe–N–C catalysts under the same/similar experimental conditions over the last five years, at least 65% higher than that of AC and 50% higher than that of commercial Pt.