SummaryDeep-sea hydrothermal vents are extreme and complex ecosystems based on a trophic chain. We are still unsure of the first colonizers of these environments and their metabolism, but they are thought to be (hyper)thermophilic autotrophs. Here we investigate whether the electric potential observed across hydrothermal chimneys could serve as an energy source for these first colonizers. Experiments were performed in a two-chamber microbial electrochemical system inoculated with deep-sea hydrothermal chimney samples, with a cathode as sole electron donor, CO2 as sole carbon source, and three different electron acceptors (nitrate, sulfate, and oxygen). After a few days of culture, all three experiments showed growth of an electrotrophic biofilm consuming directly or indirectly the electrons and producing organic compounds including acetate, glycerol, and pyruvate. The only autotrophs retrieved were members of Archaeoglobales, in all three experiments. Various heterotrophic phyla also grew through trophic interactions, with Thermococcales in all three experiments and other bacterial groups specific to each electron acceptor. This electrotrophic metabolism as energy source to drive the first microbial colonization of conductive hydrothermal chimneys was discussed.