The effect of temperature (37 and 55 °C) and inoculum-to-substrate ratios (ISRs) on biohydrogen (first-stage ISRs 0.25, 0.5, and 1) and methane (second-stage ISRs 2, 4, and 6) production during two-stage anaerobic digestion of molasses using mixed anaerobic consortia was investigated. At 37 °C, ISRs 1 and 4 produced the highest yields of biohydrogen (63.61 NL-H2/kg-VSadded) (VS: volatile solid) and methane (1092.70 NL-CH4/kg-VSadded). At 55 °C, ISR 0.5 yielded 25.03 NL-H2/kg-VSadded, while ISR 4 yielded 806.54 NL-CH4/kg-VSadded. Firmicutes, specifically Clostridia, dominated the first stage at both temperatures. The second stage (ISR 4) at 37 °C included 16.06% Chloroflexi and 22.84% Firmicutes, while at 55 °C, it included 7.55% Thermotogota.