This study was performed to investigate the influence of operational pH on dark H2 fermentation of food waste by employing anaerobic batch reactors. The highest maximum H2 yield was 1.63 mol H2/mol hexoseadded at operational pH 5.3, whereas the lowest maximum H2 yield was 0.88 mol H2/mol hexoseadded at operational pH 7.0. With decreasing operational pH values, the n-butyrate concentration tended to increase and the acetate concentration tended to decrease. The highest hydrogen conversion efficiency of 11.3% was obtained at operational pH 5.3, which was higher than that (8.3%) reported by a previous study (Kim et al. (2011) ‘Effect of initial pH independent of operational pH on hydrogen fermentation of food waste’, Bioresource Technology 102 (18), 8646–8652). The new result indicates that the dark fermentation of food waste was stable and efficient in this study. Fluorescence in situ hybridization (FISH) analysis showed that Clostridium species Cluster I accounted for 84.7 and 13.3% of total bacteria at operational pH 5.3 and pH 7.0, respectively, after 48 h operation.