Endoglucanase production in shake flasks was affected by the cultivation method, with highest production achieved using the sequential fermentation method employing pretreated sugarcane bagasse with particle sizes up to 2 mm. In cultivations using a stirred tank bioreactor, the combined effects of biomass pretreatment and agitation conditions were shown to have a strong impact on (hemi) cellulolytic enzymes production, and the advantages of the SF method and biomass pretreatment were validated. Higher enzyme production was achieved with pH control, and smaller substrate particle sizes increased the homogeneity of three-phasic STB systems, which favored endoglucanase production. These findings concerning the effects of different operational variables and cultivation methods on (hemi) cellulolytic enzymes production in a stirred tank bioreactor should contribute to the development of three-phasic cultivation systems employing different types of lignocellulosic biomass in bioreactors.