One of the current challenges to produce second-generation ethanol is to obtain an efficient enzyme complex for hydrolysis of lignocellulosic biomass. Genetic improvement of lignocellulolytic fungi has helped to obtain strains that secrete higher enzymatic titers. This article analyzes the enzymatic profile of two strains of Penicillium echinulattun, wild type 2HH and mutant S1M29, grown on different carbon sources (sugarcane bagasse pre-treated by steam explosion, cellulose, glucose, and glycerol) at different cultivation times. To determine enzyme activities, 23 substrates were used. P. echinulaturn showed potential to produce cellulases, hemicellulases, esterases, and in minor proportions, pectinases. The mutant strain showed the highest enzymatic titers to most enzymes considered and its enzymatic broth was used in the enzymatic hydrolysis of vegetable biomass. This study broadened the understanding of the enzyme complex of this fungus and the importance of future studies with the mutant strain for use in the production of second-generation ethanol.