Biofuels contribute environment-friendly, renewable energy, minimizing dependence on fossil energy. The pretreatment of biomass is a practical step that accelerates and facilitates the hydrolysis of complex materials. This work aimed to screen, select, and study cocktail formulations for biomass hydrolysis, where the enzymes were provided both from a commercial source (Trichoderma reesei, Novozymes®) and through the cultivation of Aspergillus brasiliensis and Aspergillus tamarii Kita. Experimental mixture designs were used to optimize the enzymatic conversion of substrates into simple sugars. A crude extract rich in amylase (AAB) had a significant favorable influence on cornmeal hydrolysis by maximizing the yield of reducing sugars (RS) (173 μmol mL–1). Celluclast™, rich in cellulase, significantly affected the hydrolysis of banana peel, maximizing the RS yield (175 μmol mL–1). Variable degrees of enzyme synergism were evident from statistical analysis of the biomass hydrolysis.