The microbial fuel cell (MFC) provides new opportunities for energy generation and wastewater treatment through conversion of organic matter into electricity by electrogenic bacteria. This study investigates the effect of different types and concentrations of substrates on the performance of a double chamber microbial fuel cell (DCMFC). Three mediator-less laboratory-scale DCMFCs were used in this study, which were equipped with graphite electrode and cation exchange membrane. The MFCs were fed with three different types of substrates (glucose, acetate and sucrose) at a chemical oxygen demand (COD) concentration of 1,000 mg/L. The selected substrate (acetate) was studied for three different concentrations of 500, 2,000 and 3,000 mg/L of COD. Results demonstrated that acetate was the best substrate among the three different substrates with maximum power density and COD removal of 91 mW/m2 and 77%, respectively. Concentration of 2,000 mg/L was the best concentration in terms of performance with maximum power density and COD removal of 114 mW/m2 and 79%, respectively. The polarization curve shows that ohmic losses were dominant in DCMFCs established for all three substrates and concentrations.