•The essential oil of Thymus vulgaris exhibited strong antifungal activity.•Thyme essential oil caused leakage of the cytoplasm of A. flavus.•The results indicated that this essential oil inhibited the production of aflatoxins.•The essential oil could be used as a potential source of eco-friendly fungicides.•Essential oil tested could be used to control the presence of A. flavus. The antifungal and antiaflatoxigenic properties of Thymus vulgaris essential oil (TEO) were evaluated upon Aspergillus flavus “in vitro”. Suspension containing 106 of A. flavus were cultivated with TEO in concentrations ranging from 50 to 500μg/mL. TEO reached minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) at 250μg/mL. Inhibition of ergosterol biosynthesis was detected at a concentration of 100μg/mL of TEO. Morphological evaluation performed by both light microscopy and scanning electron microscopy showed that antifungal activity of TEO could be detected starting at a concentration of 50μg/mL and the fungicide effect at a concentration of 250μg/mL. TEO completely inhibited production of both B1 and B2 aflatoxins (AFB1 and AFB2) at a concentration of 150μg/mL. This way, fungal biomass development and aflatoxin production were dependent on TEO concentration. Therefore, TEO was capable of controlling the growth of A. flavus and its production of aflatoxins.