Opuntia spp. are considered bioenergy crops for contributing against global warming while avoiding water scarcity and declining fertile land problems in their cultivation. Unfortunately, these have been scarcely researched. This work targets to evaluate the biohydrogen potential of three Opuntia varieties (Opuntia ficus-indica var. Milpa Alta, OfiM; O. ficus-indica var. Copena, OfiC; and O. engelmannii, Oeng). Hydrogen production evaluated at three inoculum-to-substrate ratios (ISR) for OfiM and OfiC, showed that the ISR decrease was inversely proportional to the maximum hydrogen cumulative production and the highest hydrogen production rate. Afterwards, hydrogen production using methanol extracted residual biomass was demonstrated feasible but resulted in a reduction in hydrogen production. The best results attained were 438 mL H2 (41 mL H2/g VS) for OfiC at ISR 0.2 The use of Opuntia species for hydrogen production should be further explored to increase its competitive potential and the possibilities of new paths on bioprocesses integration.