The treatment of residual biomass through the dark fermentation (DF) process is presented as a sustainable alternative, where bio-hydrogen is produced, which has a high energy potential, and bio-based sub-products. Consequently, this work aimed to determine the maximum organic load for the production of bio-hydrogen, from residual biomass available in Colombia. An experimental design was constructed to determine the biochemical hydrogen potential (BHP) of mixtures composed of three substrates: pig manure (PM), cocoa mucilage (CCM), and coffee mucilage (CFM). The design managed two independent variables, the organic load (10 gVS/l, 20 gVS/l, 30 gVS/l, 40 g VS/l, and 50 gVS/l), which were determined according to the physicochemical characterization of the substrates, and the S/X evaluated on two levels 1:1 and 1:2. The experiments were carried under mesophilic conditions (±35°C), a C/N ratio of 35, and a pH of 5.5 was fixed. As a result, five mixtures were evaluated, plus five targets. The reactors were erlenmeyers of 250mL, hermetically sealed, heated, and stirred in a hot plate. The tests were allowed to run until the detection of CH4; daily monitoring with a Biogas 5000® portable gas analyser did this. Following the determination of the physicochemical characteristics of the effluent, total solids (TS), volatile solids (VS), proteins, and chemical oxygen demand COD were analysed for each of the reactors. The results show that the organic load of 10gSV/L and the S/X of 1 has the highest production rate with 232 mL of H2, with a production of volatile fatty acids (VFA) of 4040mg COD/L, and a VS removal percentage of 84%. The removal of VS and the VFA allows suggesting secondary processes associated with bio-refinery schemes, for a more significant elimination of volatile solids and the obtaining of other value-added by-products.