Alternative glycerol treatment and valorization, could be a promising solution, contributing to the biodiesel production economy. Thus, the potential of electricity generation from pure glycerol, using a two-chamber microbial fuel cell (MFC), was evaluated. The effect of glycerol concentration in a range of 0.5-5.2 g COD L -1 on MFC performance was examined. In order to achieve a stable MFC performance and a high power density, special attention was paid during the acclimation phase of the anaerobic consortium, which was performed using glucose, instead of glycerol, as substrate. The best performance of the cell was observed at a glycerol concentration of 3.2 g COD L -1 . At this concentration, the Coulombic efficiency (CE) was 34.1 %, the chemical oxygen demand (COD) removal efficiency was approximately 99 % and the maximum power density was 65.4 mW m -2 . Further increase of glycerol concentration to 5.2 g COD L -1 did not enhance the MFC performance, since the power density remained at 63.4 mW m -2 , while the CE and the COD removal efficiency decreased to 22.1 and 81 %, respectively. The experimental results showed that glycerol is a suitable and promising substrate for power generation, using a simple two-chamber MFC and that acclimation of anaerobic sludge using glucose as substrate, is a suitable procedure for securing a stable MFC performance, even at high glycerol concentrations.