Monitoring parameters like Oxidation-Reduction Potential (ORP) is crucial for the intensification of the (H2) production via Dark Fermentation (DF). This study examines the initial ORP’s impact on the H2 production by DF. Batch cultures with glucose as substrate and heat-treated inoculum are conducted at various initial ORPs (−220 to −410 mV) in a 4.3 L stirred tank reactor. Real-time monitoring of pH, ORP and a bacterial analysis are performed. Metabolites composition is determined. Results indicate that initial ORPs below −250 mV enhance the process. At −284 mV, yield and productivity are 1.23 mol H2 mol−1 hexose and 32.95 mL H2 L−1 h−1, respectively, showing improvements of 89.23 and 144.57% over −220 mV. At −284 mV, lower bacterial diversity at tmax and the end correlate with higher Clostridium abundance. The optimal initial ORP range is [-284; −379 mV], comprising the ORP (−340 mV) of the maximum hydrogenase activity in the system.