This study aimed to evaluate the effect of gamma irradiation (GI) on the physicochemical, technological, antioxidant and microbiological characteristics of different whole sorghum flours (WSF), as well as to characterize the profile of chemical constituents by paper spray mass spectrometry (PS-MS). The doses applied interfered in the staining characteristics of the evaluated flours, providing the darkening of the same. For the other physicochemical, technological, and antioxidant parameters, no significant influence of the process was observed. The fingerprint obtained in both ionization modes had not been influenced by irradiation, being flavonoids, phenylpropanoids, amino acids, benzoic acid derivatives, carboxylic acids, and sugars tentatively identified. In microbiological terms, there was a reduction of molds, yeasts, and Bacillus cereus in irradiated WSF compared to control (non-irradiated). Therefore, the application of low doses of gamma irradiation represents an advantageous alternative for the conservation of WSF and maintenance of compounds bioactive identified by the PS-MS technique.