The high cost of cellulases and xylanases for the hydrolysis of lignocellulosic wastes is a bottleneck in the production of second-generation ethanol. The use of a low-cost carbon source, such as steam-exploded sugar cane bagasse (seSCB), is an alternative to reduce the costs in cellulase production. Enzyme supernatants produced by the mutant Penicillium echinulatum 9A02S1 on seSCB as a substrate have good filter paper activity (0.48 IU/mL) values combined with endoglucanases (3.24 IU/mL), beta-glucosidase (0.48 IU/mL), and xylanases (0.64 IU/mL) activities. It was found that the enzymes produced on seSCB showed activity at the same hydrolysis potential of the enzymes produced with cellulose as a carbon source. In conclusion, the results presented in this paper showed that it is possible to obtain efficient enzymes by P. echinulatum using very low-cost substrates, such as seSCB, and, therefore, contributing to the development of on-site enzyme production to produce second-generation ethanol.