Different nutritional and physicochemical conditions to obtain H2 from Citrus Peel Waste (CPW) were evaluated. For this, a screening design was carried out using Plackett and Burman design, in order to verify the main significant conditions of this process. The variables studied were pH (5.5–8.5), temperature (30–44 °C), autochthonous inoculum (0.75–2.25 gTVS.L−1), allochthonous inoculum (1–3 gTVS.L−1) and substrate (5–15 g.L−1), headspace (40–60%) and nutritional medium components, such as yeast extract (0–1 g.L−1), CaCO3, NaCl and peptone (0–5 g.L−1). The most significant operational variables were pH (8.5), allochthonous inoculum (3 gTVS.L−1) and substrate concentration (15 g CPW.L−1), conditions that favored the highest H2 (13.29 mmol.L−1) and acetic acid productions (1340 mg.L−1). Escherichia (34.5%) and Clostridium (29.93%) were the main genera identified under these conditions.