Two wild-type (WT) Aspergillus strains, A. flavus HAk1 and A. oryzae HAk2, were selected for kojic acid (KA) biosynthesis. Malt extract sucrose culture medium (MES) was the best culture medium for maximum production of KA. The maximum production of KA has been estimated at pH 4 after 7 days of incubation at 30 °C. Overproduction of KA was attained by mutagenesis of both A. flavus HAk1 and A. oryzae HAk2 through their exposer to different doses of gamma irradiation. The mutant strains (MT) A. flavus HAk1-M2 and A. oryzae HAk2-M26 were the most stable mutants for maximum production of KA through four generations. Yield of KA by A. oryzae HAk2-M26 and A. flavus HAk1-M2 has been 2.03-fold and 1.9-fold, respectively, higher than their wild-type strains. All WT and MT strains were used for KA production from different agricultural raw materials. Apple peel was the best waste for KA production by WT strains of A. flavus and A. oryzae, while orange peel and rice stalk are best material for KA production by MT strains, A. flavus HAk1-M2 and A. oryzae HAk2-M26, respectively. All experimental strains have the ability to produce considerable amounts of KA from sugarcane molasse (SCM) and sugar-beet molasse (SBM). SBM was better than SCM for KA production by all strains. The antioxidant activity of biosynthesizing KA was strongly affected with production conditions, where the highest antioxidant activity of all strains was recorded at the optimum environmental and nutritional conditions for KA production.