A glycerol-based microbial fuel cells was implemented based on activated sludge from a Wastewater Treatment Plant (WWTP). The influence of the substrate concentration was studied, observing a Monod-type behavior for the cell performance (cell voltage under closed circuit and power output) and the TSS, without improvement when the glycerol concentration in the influent exceeded 1080 mg.L-1. Under these conditions, the electrochemical performance did not rely on the substrate availability, but on the limited capability of the microorganism for electroxidating glycerol. In terms of the coulombic efficiency, half of the organic matter is degraded with simultaneous generation of electricity for a glycerol concentration of 320 mg L-1 in the influent, reducing at higher glycerol concentrations down to 20%. Finally, the anode appeared as the governing electrode, whose performance varied significantly with the glycerol concentration. © 2014 The Electrochemical Society.