Aflatoxin B1 (AFB1) is a natural carcinogen commonly present in food and feed which has deleterious effects on human and animal health. Combination treatment of microwave heating and gamma irradiation has been investigated. Artificially spiked peanuts (Arachis hypogaea L.) were used with a concentration of 300 μg/kg of AFB1 to evaluate the treatment efficacy. Qualitative and quantitative analysis of AFB1 was carried out by one dimensional thin layer chromatography and enzyme-linked immune-sorbent assay (ELISA), respectively. Gamma irradiation (5, 7 and 9 kGy) alone reduced 20 to 43% of AFB1, while, only low power microwave heating (360, 480 and 600 W) reduced it by 59 to 67%. The synergistic effect of a combination treatment at 7 and 9 kGy of irradiation at any of the chosen microwave power levels and sequence of treatment was able to reduce >95% in artificially spiked peanuts. Knowing the limitation of ELISA, an Ames test was employed to determine the mutagenicity of AFB1 in combination-treated (7 kGy and 600 W) artificially spiked peanut samples, resulting in an 80-85% reduction of its mutagenicity. Quality parameters of peanuts in terms of moisture content, water activity, hardness, colour, peroxide value and free fatty acid were evaluated and observed to be retained after combination treatments. Post-treatment sensory analysis in respect of appearance, colour, texture, taste, aftertaste and overall acceptability gave satisfactory scores. The microbiological safety of treated peanuts (exclusive and combination of 7 kGy and 360-600 W) was assessed in terms of total bacterial count, aerobic spore count, and yeast and mould count. The microbiological load was completely eliminated after exclusive gamma irradiation or the combination treatments. Combination treatment (7 kGy and 600 W) demonstrated 71 to 87% reduction in AFB1 from naturally contaminated peanuts and is recommended for further commercial application.