In this study, we investigated the effects of gamma irradiation on the antioxidant activity and metabolite profiles of Euphorbia maculata calli (PC3012). Gamma irradiation at various doses (0, 0.05, 0.5, and 10 kGy) significantly enhanced the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS⁺) radical scavenging activities of the callus extracts of PC3012 in a dose-dependent manner. High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) analyses revealed that irradiation increased the lysophospholipid content, although no new antioxidant compounds were formed. Furthermore, a PLS-DA analysis revealed evident metabolic differences between non-irradiated and irradiated samples, which were further verified by statistical validation. These findings suggest that gamma irradiation induces specific biochemical modifications that enhance the bioactive properties of PC3012 calli. This technology exhibits potential for utilization in the natural product and food sectors, particularly in the development of functional foods and nutraceuticals with improved health benefits.