The aim of this work was to assess the performances of wine byproduct biomass for hydrogen production by dark fermentation. Grape must deposits from two grape varieties (Pinot Gris and Chardonnay) were considered, either with external microbial inoculum or without. We show that grape must residues contain endogenous microflora, well adapted to their environment, which can degrade sugars (initially contained in the biomass) to hydrogen without any nutrient addition. Indeed, hydrogen production during endogenous fermentation is as efficient as with an external heat-treated inoculum (2.5 ± 0.4 LH2.L-1reactor and 1.61 ± 0.41 molH2.mol-1consumed hexose, respectively) with a lower energy cost. Hydrogen-producing bacteria were selected from the endogenous microflora during semi-batch bioreactor operation, as shown by T-RFLP profiles and 16S rRNA sequencing, with Clostridium spp. (butyricum, beijerinckii, diolis, roseum) identified as the major phylotype. Such hydrogen production efficiency opens new perspectives for innovating in the valorization of winery by-products.