AbstractMycotoxin-producing molds which considered as common maize grains contaminants are the genera Fusarium, Aspergillus and Penicillium. There are natural and safe ways to protect grains from mold contamination as the use of essential oils and chemical treatments. A total number of 25 samples were used to study the natural frequency in five governorates in Egypt, Molecular identification indicated that the most frequent fungi were Fusarium verticillioides, Aspergillus niger, Talaromyces verruculosus, Aspergillus flavus and Aspergillus terreus. The in vitro studies have been done to determine mycelial growth and spore germination inhibition of the two A. flavus; isolated and reference isolates. Thyme and acetic acid were tested in direct contact assay to study their effects on mycelial growth. Treatments showed significant impact on mycelial growth and spore germination inhibition of both A. flavus isolates. In the postharvest application treatments: as vapour and carrier contact assay, Thyme and Acetic acid were tested to determine their influence on growth and aflatoxin production in A. flavus isolates by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Results indicated that both treatments were effective in inhibition of aflatoxin production in both vapour and carrier assays as they succeeded in reducing AFB1 while they inhibited completely the production of AFB2. The extent of the inhibition of aflatoxin production was dependent on the concentration and storage duration of treatments applied.