ASPERGILLUS flavus produces many secondary metabolites including aflatoxin B1, the most toxic and most potent carcinogenic natural compound that causes contamination on a variety of food and feed commodities. The aim of this study was to investigate the effect of Lactobacillus rhamnosus, the essential oils anise, caraway, fennel and potassium sorbate on growth rate, and aflatoxin B1 accumulation, and also to measure the expression of aflR a regulatory gene of aflatoxin biosynthesis pathway by using real time-q PCR technique. Our results indicated that all treatments exhibited potential antiaflatoxigenic and antifungal effect against A.flavus. Furthermore, L. rhamnosus after 7days of incubation, anise oil at concentration 100µl/100ml medium, and potassium sorbate at concentration 4.0µg /100ml medium at pH 6.0 showed a lowered transcription level of aflR gene as compared to control. The results indicated that L. rhamnosus may be useful as a natural biocontrol and strengthens the utilization of anise oil as an ideal antimicrobial against A.flavus. Visually, the results of real time-q PCR technique for aflR gene expression indicated that it was not a useful method for diagnosis of non-aflatoxin producing strains or vise viscera.