This research aims to provide insights into the biological efficacy of a newly formed hydrophobic deep eutectic solvent (DES). A DES based on menthol was successfully synthesized with fatty acids. The DESs� properties as enzyme activators were examined against a neat counterpart. The menthol:decanoic acid (1:1) combination showed improved thermal stability, strong catalytic activity, and reusability for up to four subsequent cycles under ideal conditions (pH 7.0, 40 °C for 2 h). The hydrophobic DES replaced hexane in ester synthesis, where RNL@DES5 showed better fatty acid conversion compared to neat RNL. This study demonstrated promising applications of hydrophobic DESs in non-aqueous organic reactions. © 2023 by the authors.