The oxidation-reduction potential (ORP) is an important factor in H2 production via dark fermentation however its effect over microbial diversity in an acidogenic sludge has not, been well studied. This work studies the effect of ORP controlled by hydrogen peroxide and potassium ferricyanide on continuous hydrogen production and microbial diversity in an acidogenic sludge fed (HRT 12 h and pH 5.5) with glycerol. Results show that the more oxidizing ORP control environment (−540 mV) improves H2 yield by 50–70% (0.31–0.51 molH2/mol glycerol) over non-ORP control conditions. Oxidizing ORP values were shown to enrich microorganisms of the genus Clostridium, which have been linked to high H2 yields. Therefore, controlling ORP in an acidogenic sludge was shown to directly modify microbial diversity at the genus level, and could likely to indirectly regulate metabolic function. Additionally, metabolic pathways were regulated by the kind of agent used.