The individual and mutual effects of substrate concentration (from 0.8 to 9.2 g/L) and pH (from 4.6 to 7.4) on hydrogen and volatile fatty acids production from sugarcane bagasse (SCB) were investigated in batch reactors, using a response surface methodology (RSM) and central composite design (CCD). The maximum of 23.10 mmoL H2/L was obtained under optimized conditions of 7.0 g SCB/L and pH 7.2, at 37 °C through the acetic acid pathway (1.57 g/L). Butyric and succinic acids were the major volatile fatty acids (VFA) produced in the fermentation process (from 0.66 to 1.88 g/L and from 1.06 to 1.65 g/L, respectively). According to the results, the RSM and CCD were useful tools to achieve high hydrogen production rates using Clostridium, Bacillus and Enterobacter, identified by Illumina sequencing (16S RNAr) in the fermentative consortium, and Clostridium and Paenibacillus, autochthonous bacteria from SCB. Significant changes were observed in the microbial community according to the changes in the independent variables, since the genera in the central point condition (5.0 g SCB/L and pH 6.0) were Lactobacillus, Escherichia and Clostridium, and Bacteroides and Enterobacter, which were identified in the optimized condition (7.0 g SCB/L and pH 7.2).