In Mexico, the industrial production of tequila leads to the discharge of more than 31.2 million of m3 of vinasse, which causes serious environmental issues because of its acidity, high organic load and the presence of recalcitrant compounds. The aim of this research was to study the feasibility of a fixed bed reactor for the production of biohydrogen by using tequila vinasse as substrate. The experiments were carried out in a continuous mode under mesophilic and acidic conditions. The maximum hydrogen yield and hydrogen production rate were 1.3 mol H2 mol/mol glucose and 72 ± 9 mL H2/(Lreactor h), respectively. Biogas consisted of carbon dioxide (36%) and hydrogen (64%); moreover methane was not observed. The electron-equivalent mass balance fitted satisfactorily (sink of electrons from 0.8 to 7.6%). For vinasses, hydrogen production accounted for 10.9% of the total available electron-equivalents. In the liquid phase, the principal metabolites identified were acetic, butyric and iso-butyric acids, which indicated a butyrate–acetate type fermentation. Tequila vinasses did not result in potential inhibition of the fermentative process. Considering the process as a water treatment system, only 20% of the original carbon was removed (as carbon dioxide and biomass) when the tequila vinasses are used.