Dark fermentation using mixed cultures is a promising biotechnology for producing hydrogen (H2) from renewable organic waste at a low cost. The impact of the characteristics of carbohydrates was evaluated on H2 production and the associated changes in clostridial populations. A series of H2-producing batch experiments was performed from mono-, di- to tri-saccharides (i.e. fructose, glucose, sucrose, maltose, cellobiose, maltotriose). Both chain length and alpha- or beta-linkage of carbohydrates impacted H2 production performance as well as the patterns of hydrogenases. The H2 yield, ranging from 1.38 to 1.84 mol-H2/mol-hexose, decreased with the increasing chain length of the carbohydrates, showing a negative effect of the hydrolysis step on H2 production efficiency. Changes in H2 yield were associated with a specialization of clostridial species, which used different metabolic routes. The rise in H2 production was associated with butyrate and acetate increases while H2 consumption was related to caproate formation. Both clostridial [FeFe]- and [NiFe]-hydrogenases were identified in cellobiose cultures by a proteomic approach. This is the first study that combines genetic and proteomic analyses focused on H2-producing bacteria under various conditions and it opens very interesting perspectives to better understand and optimize H2 production using mixed cultures.