Electro-fermentation has been recently proposed as a new operational mode of bioprocess control using polarized electrodes. This paper aims to evaluate how polarized electrodes are affecting microbial metabolic fermentative pathways, with a special focus on how the bacterial populations are affected during hydrogen production by dark fermentation. Four different potentials were applied on the working electrode in batch electro-fermentation tests operated with mixed culture and using glucose as a substrate. Two different metabolic behaviours for H2 production were observed in electro-fermentation. The first one led to a higher H2 production compared to conventional fermentation with a strong selection of Clostridium sp. The second behaviour led to lower H2 production along with ethanol, and strongly correlated with the selection of Escherichia and Enterobacter genera. However, the effect of the applied potential on population selection was mostly non-linear and no simple relationship was found between these two parameters. Overall, electro-fermentation process has shown its potential as a new type of control for mixed-culture bioprocesses with significant effects of polarized electrodes on glucose fermentation.