Aspergillus flavus and A. parasiticus are commonly found in nut-growing areas in California. Some isolates of these species produce aflatoxins that very occasionally contaminate nut kernels. The A. flavus strain AF36, which does not produce aflatoxins, is registered as a biocontrol for use in tree nuts in California. AF36 success in aflatoxin reduction relies on the capacity to displace the native toxigenic isolates. Currently, the biocontrol AF36 is quantified by vegetative compatibility group analysis, which is expensive and time consuming. We developed a quantitative real-time PCR (qPCR) procedure to quantify the proportions of both AF36 and the toxigenic isolates at reduced time and assay costs. Specific primers to target the AF36 strain, and A. flavus and A. parasiticus were designed. The standard curves were generated to quantify the amounts of DNA based on the threshold values (Ct) for each strain of interest. Verification tests showed significant correlations between known concentration of spore mixtures and the amounts quantified by the qPCR assay. The qPCR assay was used to quantify the proportions of AF36 from leaves, nuts, and soil samples, demonstrating its usefulness to accurately quantify AF36 and the natural strains of the pathogen. This study demonstrates the potential of qPCR assay to increase the number of samples that can be quantified based on available resources and on time and improve the study of the epidemiology of aflatoxin producing fungi.