Essential oils (EOs) are complex mixtures of compounds derived from plants that exhibit antimicrobial activity. Several studies have demonstrated their antifungal activity in food matrices or in vitro via vapor phase or direct addition. Recently, researchers are focusing on elucidating the target site or the mechanism of action of various EOs. Past research has suggested evidence of how EOs act in the fungal cells via assays assessed from cell wall alterations or gene expression modifications. However, no previous reports have summarized most methods for finding the target site of the mechanism of action for EOs. Therefore, this review presents the methods and assays used to discover the target site or the mechanism of action of EOs against fungal cells. Researchers commonly analyze the plasma membrane integrity using various techniques as well as the changes in cell morphology. Meanwhile, the quantification of the activity of the mitochondrial enzymes, ROS species, and gene expression are less assayed.

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