Summary

Essential oils (EOs) from the leaves of Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens, representing three chemotypes—thymol-rich, carvacrol-rich, and a mixed thymol–carvacrol type—were chemically characterized and comparatively assessed for their antifungal, insecticidal, and enzyme-inhibitory activities. This integrated approach provides a comparative assessment of all three chemotypes across multiple biological models, including phytopathogenic fungi, insect bioassays, and key enzyme targets. All EOs displayed antifungal activity for the tested phytopathogenic fungi (Alternaria alternata, Botrytis cinerea, and Fusarium oxysporum) at concentrations above 0.5 mg/mL, with the thymol-rich chemotype showing the highest activity. The minimum inhibition concentration for Oidium farinosum conidial growth was determined and found to be similar for thymol and carvacrol chemotypes and lower for the terpene mixture. Insect control activity was evaluated by an antifeeding assay, where carvacrol and especially thymol chemotypes can be classified as feeding deterrents. EOs and standards revealed a weak toxicity against Ceratitis capitata, with less than 20% mortality at a concentration of 50 mg/mL, and both chemotypes were found to be ineffective in preventing egg deposition. The acetylcholinesterase (AChE) inhibition assay revealed that carvacrol had the greatest inhibitory effect on AChE, followed by EOs, and, finally, thymol. Regarding the α- and β-glucosidase (α- and β-GLU) inhibitory assays, thymol had the strongest inhibitory effect on α-GLU, while plant β-GLU was not inhibited by the standards or OEs.