THIS STUDY aimed to the potency of two species of Lactobacillus (LAB) i.e. Lactobacillus rhamnosus, L.bulgaricus and Streptococcus thermophilus in inhibition of A. flavus growth, production of aflatoxin B1and expression of the aflR gene of aflatoxin biosynthesis pathway by using real time-q PCR technique. The trail was designed in two variations challenging the fungi with seven treatments of LAB species and S. thermophilus, before and at same time (simultaneous) fungal inoculation The results indicated that all treatments exhibited potential antiaflatoxigenic and antifungal effect against A. flavus. After 72h of incubation aflatoxin B1 not detected with some species treatments compared to control which produced aflatoxin B1 1840±20µg /100 ml medium. Furthermore, the results showed that detection of transcription level of aflR gene was not correlated to the actual toxicity of each treatment. The mRNA abundances of aflR gene in control was 1± 0.11, while in treatments with (L. rhamnosus; S. thermophilus; L. rhamnosus & S. thermophilus; L. bulgaricus & L. rhamnosus; L.bulgaricus & S. thermophilus and L. rhamnosus & L. bulgaricus & S. thermophilus) were 3.567± 0.25, 1.564± 0.13, 0.421± 0.05, 0.767±0. 06,0.585± 0.05 and 1.498± 0.12, respectively. These differences in gene expression profiles suggested that there was specificity between gene response and treatment. Finally, the results of real time-q PCR technique for aflR gene expression indicated that it was in appropriate method for diagnosis aflatoxin producing and non- producing strains