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The search for renewable and sustainable sources of energy has been one of the main goals of society in recent years, especially to reduce the environmental impacts of fossil fuels. One promising alternative is the production of hydrogen, which does not emit greenhouse gases and can be produced from agro-industrial wastes. The Clostridium genus is recorded as having high hydrogen yields compared to other genus, with several producing species. The objective of this work was to evaluate biohydrogen production potential of four agro-industrial residues, which were soft drink wastewater, corn steep liquor, cheese whey, and expired Guaraná soft drink, using one model strain Clostridium beijerinckii ATCC 8260 and newly isolated Clostridium butyricum DEBB-B348. The agro-industrial wastes were characterised in terms of monosaccharide, organic acid, amino acid, cation, and anion concentrations and compared to the literature. 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 an R2 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. The C. butyricum 16s rRNA gene phylogenetic tree and the carbohydrate, organic acid, and amino acid kinetics of the optimum medium are also presented. These results indicate a potential hydrogen production process utilising less expensive substrates, proposing more proper disposal for agro-industrial wastes and using an isolated strain with high yield.