Abstract: Neutral red and potassium ferricyanide are two electron mediators that can increase the efficiency of microbial fuel cells (MFCs) via significantly different mechanisms. In this study, effects of the two mediators on electricity generation and microbial structure of a toluene powered MFC was first investigated. The maximum voltage (Vmax) was 110.4 mV and the half-saturation constant (Ks) was 12.8 mg/L in the absence of mediators. Although the power generation efficiency was increased when adding modest amount of mediators to the anode, the toluene degradation time was 1.56–2.15 times longer than that of unmediated MFC’s, relying on the mediator used. Denaturing gradient gel electrophoresis (DGGE) analysis showed a mediator-related characteristic of the microbial structure in the MFC. Results of this investigation can be used as a basis for future assessment and design of MFCs powered by xenobiotics-contaminated wastewater, such as toluene.