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This study reported the impact of electron beam (e-beam) treatment on microbiota and mycotoxins naturally present in red pepper powder and physicochemical quality changes. Treatment at 6 kGy indicated significant (p < 0.05) decontamination of yeasts and molds by 3.0 and 4.4 log CFU/g, respectively. A reduction of 4.5 log CFU/g of total plate counts (TPC) was observed at 10 kGy for 23 s. Fungal inactivation followed first-order kinetics while TPC better fitted with Gompertz function (R2 = 0.9912). E-beam treatment was not efficient for the degradation of aflatoxins but indirectly controlled their production by inactivation of mycotoxigenic molds. Indeed, reduction of 25% ochratoxin A was recorded at 30 kGy retaining >85% of total phenols, carotenoids and antioxidants activity. Moreover, treatment impact on total color difference (ΔE*) indicated ‘slight differences’. Overall, e-beam treatments up to 10 kGy were efficient in decontaminating the natural microbiota without detrimental effects on the physicochemical qualities of red pepper powder.