This study aimed to evaluate the capacity of different inoculum sources and their bacterial diversity to generate hydrogen (H2). The highest Simpson (0.7901) and Shannon (1.581) diversity indexes for H2-producing bacterial isolates were estimated for sewage inocula. The maximum cumulative H2 production (Hmax) was 639.6 ± 5.49 mL/L recorded for the sewage inoculum (SS30) after 72 h. The highest H2-producing isolates were recovered from SS30 and identified as Clostridium saccharobutylicum MH206 and Lactobacillus brevis MH223. The Hmax of C. saccharobutylicum, L. brevis, and synergistic coculture was 415.00 ± 24.68, 491.67 ± 15.90, and 617.67 ± 3.93 mL/L, respectively. The optimization process showed that the Hmax (1571.66 ± 33.71 mL/L) with a production rate of 58.02 mL/L/h and lag phase of 19.33 h was achieved by the synergistic coculture grown on 3% molasses at 40 °C, pH 7, and an inoculum size of 25% (v/v). This study revealed the economic feasibility of the synergistic effects of coculture on waste management and biohydrogen production technology.