The objective of this study was to optimize the enzymatic pretreatment process of extracting menthol, a major component of the essential oil stored in the trichomes of fresh leaves of Mentha arvensis, by the action of the crude enzymatic extract previously produced by the Aspergillus niger fungus cultivated in cactus pear. Under the experimental conditions of 42 degrees C, 141 min and 58 mL of enzyme in 442 mL of water, there was an increase of 186.63% in the hydrodistillation yield, which represents an acceptable positive deviation of 12.54% compared to the predicted value of the model obtained by experimental design. Trichome rupture can be confirmed by scanning electron microscopy (SEM) and the chemical structure of the obtained menthol was not modified according to gas chromatography and infrared spectroscopy. The enzymatic pretreatment applied before the hydrodistillation step was successful and proves the potential of the association of biotechnological techniques with a physicochemical process.