Abstract At high electrical current regime power overshoot may occur in microbial fuel cell (MFC) operation with both cell voltage and electrical current declining at reduced external loads. This study demonstrated the power overshoot in a two-chamber MFC using acetate and oxygen respectively as anodic and cathodic fuels. The cell worked well until reaching 0.51 V and 790 mA/m 2 at power density of 400 mW/m 2; further reducing external load leads to decrease in both cell voltage and generated current. During the noted power overshoot regime the internal resistance of MFC increased monotonically with decreasing external load. Based on the electrochemical analysis of anodic and cathodic losses, the occurrence of power overshoot is proposed to be determined by the combined resistance of intracellular loss and of extracellular electron transfer (EET) loss.