Abstract: Wild type strain Bacillus subtilis UTB1 isolated from Iran had shown antagonisticactivity against Aspergillus flavus in pistachio. In a previous study, we found that this strainproduces lipopeptides and is able to degrade Aflatoxin B1, a toxin produced by A. flavus. In thiswork, we are interesting in improving the antagonistic activity of the strain UTB1 againstA. flavus. To do so, we designed a random mutagenesis of UBT1 using gamma irradiation. Weevaluated the effects of different doses of irradiation (from 100Gray to 3000Gray) and studied500 colonies against A. flavus in dual culture assay. We found that 45 colonies exhibited higherinhibition activity compared to the non-irradiated wild type. Then we selected six mutants out ofthe 45 based on the different polymorphism patterns obtained by rep-PCR (ERIC and BOX). Thesix strains demonstrated significant differences with wild type UTB1 in terms of production ofbiosurfactants and also swarming motility. TLC chromatograms have shown overproduction oflipopeptides surfactin, fengycin and iturin compared to the wild type. These results suggest thatthese strains would be promising biocontrol candidates against of A. flavus in pistachio.