The technical feasibility of valorizing tequila vinasse (TV), a wastewater with high pollution potential, through the production of biogenic hydrogen via dark fermentation, has long been proven in diverse lab-scale reactors that were operated either in batch or continuous mode. However, such systems have mainly been tested with diluted streams and nutrient supplementation, hindering the techno-economic attractiveness of the TV-to-hydrogen concept at large scale. In this study, the feasibility of producing hydrogen from high-strength undiluted TV with no added extra nutrients was evaluated under batch mesophilic conditions. Additionally, the use of two different acidogenic inocula obtained either by heat or heat-aeration pretreatment was investigated to get a greater understanding of the effect of inoculum type on the process. The results obtained showed that the TV utilized herein contained macro- and micro-nutrients high enough to support the hydrogenogenic activity of both cultures, entailing average hydrogen yields of 2.4–2.6 NL H2/L vinasse and maximum hydrogen production rates of 1.4–1.9 NL H2/L-d. Interestingly, the consumption of lactate and acetate with the concomitant production of butyrate was observed as the main hydrogen-producing route regardless of the inoculum, pointing out the relevance of the lactate-driven dark fermentative process. Clostridium beijerinckii was ascertained as key bacteria, but only in association with microorganisms belonging to the genera Enterobacter and Klebsiella, as revealed by phylogenetic analyses.