This paper investigates the biohydrogen production by two mesophilic strains, a strict anaerobe (Clostridium butyricum CWBI1009) and a facultative anaerobe (Citrobacter freundii CWBI952). They were cultured in pure and mixed cultures in serum bottles with five different carbon sources. The hydrogen yields of pure C. freundii cultures ranged from 0.09 molH2.molhexose-1 (with sucrose) to 0.24 molH2.molhexose-1 (with glucose). Higher yields were obtained by the pure cultures of Cl. butyricum ranging from 0.44 molH2.molhexose-1 (with sucrose) to 0.69 molH2.molhexose-1 (with lactose). This strain also fermented starch whereas C. freundii did not. However, it consumed the other substrates faster and produced hydrogen earlier than Cl. butyricum. This ability has been used to promote the growth conditions of Cl. butyricum in co-culture with C. freundii, since Cl. butyricum is extremely sensitive to the presence of oxygen which strongly inhibits H2 production. This approach could avoid the addition of any expensive reducing agents in the culture media such as L-cysteine since C. freundii consumes the residual oxygen. Thereafter, co-cultures with glucose and starch were investigated: hydrogen yields decreased from 0.53 molH2.molhexose-1 for pure Cl. butyricum cultures to 0.38 molH2.molhexose -1 for mixed culture with glucose but slightly increased with starch (respectively 0.69 and 0.73 molH2.molhexose-1). After 48 h of fermentation, metabolites analysis confirmed with microbial observation, revealed that the cell concentration of C. freundii dramatically decreased or was strongly inhibited by the development of Cl. butyricum.