Single chamber air-cathode microbial fuel cells (MFCs) were operated for 6 months to demonstrate the impact of biofouling distribution on cathode performance. Total biofouling decreased the maximum power density by 38% from 892 ± 8 mW m⁻² to 549 ± 16 mW m⁻². Cleaning surface biofouling slightly enhanced the power density by 12%, but additional removing of the biofouling inside the catalyst layer further increased power output by 30% to 802 ± 14 mW m⁻², indicating that inner biofouling aggressively inhibits cathode activity. Compared with surface biofouling, the inner biofouling clogged a portion of pores in the catalyst layer, which severely reduced oxygen permeability, conductivity and reaction sites. Consequently, the kinetic activity of the cathode was impaired as the exchange current density declined and the charge transfer resistance increased. Thus, it is shown that the biofouling within the catalyst layer plays a more crucial role for air cathodes over long-term operation.