Carvacrol (5-Isopropyl-2-methylphenol), a volatile oil constituent, mainly exists in Labiaceae family plants. Carvacrol has long been studied for its natural antifungal potential and food preservative potential. However, its exact mode of action, especially against Penicillium digitatum (P. digitatum), remains unexplored. Herein, a 1 H-NMR-based metabolomic technique was used to investigate the antifungal mechanism of carvacrol against P. digitatum. The metabolomic profiling data showed that alanine, aspartate, glutamate, and glutathione metabolism were imbalanced in the fungal hyphae. A strong positive correlation was seen between aspartate, glutamate, alanine, and glutamine, with a negative correlation among glutathione and lactate. These metabolic changes revealed that carvacrol-induced oxidative stress had disturbed the energy production and amino acid metabolism of P. digitatum. The current study will improve the understanding of the metabolic changes posed by plant-based fungicides in order to control citrus fruit green mold caused by P. digitatum. Moreover, the study will provide a certain experimental and theoretical basis for the development of novel citrus fruit preservatives.aroused by excessive use of certain routine and synthetic antifungal agents. Over the past few decades, the intentions have been to ensure the hygiene and safety levels of fruit products along with promoting maximum biodegradability. In order to achieve this ambition, researchers are still trying to screen natural products with strong antifungal potential from plants. Moreover, it has also been tried to explore new preservation technologies that can replace synthetic fungicides. Compared with synthetic fungicides, natural antifungal substances have attained much attention in recent years [6][7][8][9].Various essential oils refined from Melaleuca alternifolia have strong inhibitory effects on multiple fungal strains, particularly P. italicum Wehmer and P. digitatum Sacc., and hence can be used as natural preservatives in agricultural and food processing [10]. Previously we have reported that cinnamaldehyde contained in cinnamon has a good antifungal effect against P. italicum and clarifies the method for dynamic detection of the antifungal effect of cinnamaldehyde [11]. In addition, GC-MS and GC-FID techniques successfully separated the essential oils components viz. limonene (87.02%), linalyl acetate (53.76%) and linalool (39.74%) from peels, leaves and flowers of Citrus aurantium var amara, neroli oil appeared as the best fungistatic agent, which reduced the P. digitatum sporulation to 25 % at a 50 mg/mL concentration of oil [12].Carvacrol (5-Isopropyl-2-methylphenol) is the major volatile oil constituent from Labiaceae family plants, such as oregano, mint, and thyme [13]. Carvacrol has many medicinal and edible applications, including antifungal, antimicrobial, anti-inflammatory, insecticidal, antitumor, and other effects [14]. Carvacrol inhibited the growth of P. digitatum and P. italicum in in vitro and in vivo experiments of l...