Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method.