Most microbial electrosynthesis systems depend heavily on the availability of hydrogen as electron carrier. To improve hydrogen availability in microbial processes, in situ hydrogen production is required with a catalyst that is compatible under microbial conditions (near-neutral pH, mesophilic temperature, aquous electrolyte). Here, we demonstrate the use of trace metal compounds from microbial medium as hydrogen evolution reaction (HER) electro-catalyst under microbial compatible conditions. Concentrated mixtures (10 vol%) of the metal compounds present in the microbial medium (trace metal mix medium; containing Co, Cu, Fe, Mn, Mo, Ni and Zn salts and ethylenediaminetetraacetic acid (EDTA)) were added to electrochemical reactors (controlled at -1.06 V vs Ag/AgCl and flushed with CO2/N2). After addition of the concentrated trace metal mix, the cathodic current increased up to 15 times with high electron recovery into hydrogen (70–100%). The formed HER catalyst performance was also measured in microbial growth medium (with ammonium and 0.1 vol% trace metal mix), showing similar performance rates compared to the concentrated trace metal mix medium. Further identification of the active compounds within the mix emphasized the role of Cu and Mo, a mix with Cu and Mo showed the same catalyst performance as the trace metal mix with all previously mentioned compounds. Moreover, the performance of the trace metal mix without EDTA showed the highest increased current (7 times higher up to -240 A/m2 or -80 kA/m3), decreased hydrogen overpotential (55 mV at -10 A/m2) and increased exchange current density (1.36 mA/m2). Integration of the discovered HER catalyst in biological systems will allow in situ hydrogen production in bio-electrochemical and fermentation systems.