H2 and ethanol production from glycerol-containing wastes discharged from a biodiesel fuel production plant by Enterobacter aerogenes NBRC 12010 was demonstrated in bioelectrochemical cells. Thionine as an exogenous electron transfer mediator was reduced by E. aerogenes, and was re-oxidized by a working electrode applied at +0.2 V against a Ag/AgCl reference electrode by a potentiostat (electrode system). At the initial glycerol concentration of 110 mM, 92.9 mM glycerol was consumed in the electrode system with 2 mM thionine after 48 h. On the other hand, the concentration of glycerol consumed was only 50.3 mM under the control conditions without thionine and the electrodes (normal fermentation). There are no differences in the yields of H2 and ethanol against glycerol consumed between the control conditions and the conditions with the electrode system. A pH of 6.0 was suitable for the H2 production in the range between pH 6 and pH 7.5 in the electrode system. At pH values of 7.0 and 7.5, H2 production decreased and formate was remarkably produced in the reaction solution. The rates of both glycerol consumption and the H2 and ethanol production increased as the thionine concentration and the surface area of the working electrode increased. After 60 h, 154 mM of the initial 161 mM glycerol concentration in the wastes was consumed in the electrode system, which is a 2.6-fold increase compared to the control experiment. Biotechnol. Bioeng. 2007;98: 340–348. © 2007 Wiley Periodicals, Inc.