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Leaves of Croton argyrophyllus contain essential oil with promising active components for the development of drugs and botanical insecticides. In this study, we evaluated the enzymatic pretreatment process to increase the extraction of essential oil from fresh and dried leaves of C. argyrophyllus. Pretreatment was carried out using a crude multienzymatic extract obtained via solid‐state fermentation of forage palm by Aspergillus niger, and the extraction was performed by hydrodistillation. A Doehlert matrix was used to optimize the enzymatic pretreatment variables temperature and enzymatic extract. The effect of pretreatment time was also investigated. At optimum experimental conditions, 41.34°C, 140 min, and 130.73 mL of enzyme in 369.27 mL of water, the essential oil yield from fresh leaves subjected to enzymatic pretreatment increased by 9.35% and that from dry leaves by 6.77%. Based on chromatographic analysis (GC–MS), no compound was degraded in the extraction process. Micromorphological analysis confirmed the rupture of the glandular trichomes, favoring essential oil release. Therefore, enzymatic pretreatment associated with hydrodistillation increased the essential oil yield and is a promising application to obtain essential oil for therapeutic purposes without altering its composition.

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