The objective of this work was to evaluate the potential of biohydrogen production in agro-industrial residues, which were soft drink wastewater (SDW), corn steep liquor (CSL), cheese whey (CW), and expired Guarand soft drink, using one model strain Clostridium beijerinckii ATCC 8260 and newly isolated Clostridium butyricum DEBB-B348. The agro-industrial residues were characterized, and all of them contained significant concentrations of carbohydrates such as glucose, fructose, lactose, and maltose, and in the case of CSL and CW they also contained significant concentrations of amino acids. After performing subsequent experimental designs, the significant factors were cheese whey concentration, corn steep liquor concentration, and fermentation time for C. beijerinckii, and corn steep liquor concentration and fermentation time for C. butyricum (p < 0.05), with R-2 of 0.950 and 0.895, respectively. The maximum hydrogen volume production was 18.5 +/- 1.68 mL and 27.4 +/- 1.84 mL for each strain, respectively. It is noteworthy the high yield of hydrogen produced with C. beijerinckii (235 mL H-2/g(CODremoved)) and C. butyricum (310 mL H-2/g(CODremoved)). It was also noted that CSL and/or CW-based culture media do not need to be supplemented with cysteine-HCl. These results indicate a potential hydrogen production process utilizing less expensive substrates.