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<i>Aspergillus flavus</i> is a common contaminant in grain, oil and their products. Its metabolite aflatoxin B₁ (AFB₁) has been proved to be highly carcinogenic. Therefore, it is of great importance to find possible antifungal substances to inhibit the growth and toxin production of <i>Aspergillus flavus</i>. Carvacrol (CV) was reported as a potent antifungal monoterpene derived from plants. In this paper, the antifungal effects and mechanism of CV on <i>Aspergillus flavus</i> were investigated. CV was shown good inhibition on the growth of <i>Aspergillus flavus</i> and the production of AFB₁. CV used in concentrations ranging from 0, 50, 100 and 200 μg/mL inhibited the germination of spores, mycelia growth and AFB₁ production dose-dependently. To explore the antifungal mechanism of CV on <i>Aspergillus flavus</i>, we also detected the ergosterol content of <i>Aspergillus flavus</i> mycelia, employed Scanning Electron Microscopy (SEM) to observe mycelia morphology and utilized Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS) to explore the lipidome profiles of <i>Aspergillus flavus</i>. The results showed that the production of ergosterol of mycelia was reduced as the CV treatment concentration increased. SEM photographs demonstrated a rough surface and a reduction in the thickness of hyphae in <i>Aspergillus flavus</i> treated with CV (200 µg/mL). In positive ion mode, 21 lipids of <i>Aspergillus flavus</i> mycelium were downregulated, and 11 lipids were upregulated after treatment with 200-µg/mL CV. In negative ion mode, nine lipids of <i>Aspergillus flavus</i> mycelium were downregulated, and seven lipids upregulated after treatment with 200-µg/mL CV. In addition, the analysis of different lipid metabolic pathways between the control and 200-µg/mL CV-treated groups demonstrated that glycerophospholipid metabolism was the most enriched pathway related to CV treatment.