The substrate and yeast extract (YE) concentrations of a batch reactor were changed according to a central composite design in order to optimize the conversion of sugarcane bagasse (SCB) into hydrogen. The optimum hydrogen production (1.50 mmol/L) was obtained using 2.77 and 5.84 g/L of YE and SCB, respectively. Taxonomic analysis using the SEED database indicated that Clostridium (33% of total communities) and Methanothermobacter (40% of archaeal community) were the most abundant genera in the high hydrogen performance reactor. Key microorganisms and related pathways involved in all steps of the anaerobic digestion were further revealed and may help drive sugarcane bagasse bioconversion.