The second generation (2G) ethanol process has been improved in the last decades, which allowed 2G ethanol production in industrial scale. However, the cellulolytic enzyme cocktail (CEC) usage remains one of the most important operational costs. An alternative to enzyme cost reduction is on-site manufacturing using high CEC productivity fungi and enzymes with high conversion capacity. This study aimed to evaluate Trichoderma harzianum CEC production in 1.5 L working volume fermentor under different agitation rate conditions and impeller type (Rushton and Pitched blade) as first step to determine a scale-up criteria to be tested in pilot scale fermentor. The results showed no statistically significant difference for the agitation range and impeller type conditions. Further experiments tested P49P11 T. harzianum wild strain against a low foam T. harzianum mutant strain, 5PCE15. The mutant strain showed an equivalent performance with the advantage of no foam formation. These results are decisive for further experiments aiming a robust process scale-up.