In the present investigation, the effects of the substrate composition, organic load, medium acidification time, operation pH, and temperature on the production of hydrogen by anaerobic fermentation with fruits and vegetable waste, and fresh mucilage of coffee was evaluated. For this purpose, tests were carried out in a 20-liter bioreactor operated in batch mode, under a central composite experimental design (CCD). The fermentations were conducted under mesophilic conditions, without adding inoculum, and without sterilizing the substrate. The results for maximum daily hydrogen production (MDP), the maximum hydrogen content in the gas (MHC) and cumulative production (CHP) showed an acceptable fit to second-order polynomial models. All the independent variables were significant, especially the operation pH and the acidification time. Also, the premises for a model obtained by regression, according to error analysis, were fulfilled. In the same way, it was possible to optimize the response variables. The maximum specific production concerning the organic load was 5511 mL H2 gCODremoved-1, and regarding the volatile solids was of 670 mL H2 gVSadded-1. These values are higher than those reported with similar substrates in continuous fermentation, with cell retention, use of inoculum, and substrate pretreatment.