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The main objective of this research is to assess how gamma radiation influences the chemical composition and activities such as antioxidant, antimicrobial and anticancer activities of Lepidium sativum(L. sativum) seeds. Methanolic extracts were obtained through a 24 h maceration process from L. sativumseeds exposed to various doses of gamma irradiation. Phytochemical screening involved precipitation and staining reactions, with the antioxidant potential assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Antimicrobial activity was evaluated through a diffusion test on agar medium, while the cytotoxic potential on the lung cancer line “A-549 cells” was examined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Qualitative analysis of the extracts identified preserved bioactive constituents after irradiation. Additionally, quantitative analysis indicated a significant rise in the total content of polyphenols and flavonoids under radiation, accompanied by an enhanced reducing power compared to the control. The antimicrobial and cytotoxic capabilities also improved, evident in increased growth inhibition zones for certain microbial strains and reduced viability rates of A549 cells. These results show promise and warrant further investigation into the potential of gamma radiation to enhance additional biological effects of plants.