The energy output of a dual chamber anoxic microbial fuel cell (MFC) subjected to variation in the substrate concentration was investigated. Wastewater from a primary clarifier effluent stream was used as the MFC inoculum. The power densities and efficiencies of the respective MFC operating cycles were obtained and analysed. The microbial growth in the anodic chamber was also quantitatively described via absorbance readings. A direct correlation between the closed-loop voltage output, substrate concentration and microbes were observed. The maximum voltage output increased with increasing substrate concentration from 66.6 mV at 2.78 mM to 96.4 mV at 5.56 mM, and this correlated with an increase in the microbial cells. Further increase in substrate concentration resulted in a decrease in the closed-loop voltage output by 46 % to 51.9 mV at 27.78 mM, with maximum power densities of 10.42 mW.m-2. The decrease in voltage output is attributed to substrate inhibition. Secondly, at very low pH or acidic condition, microbial activities and metabolism was inhibited.