Selenium nanoparticles (SeNPs) have attracted considerable interest for their promising biomedical applications, particularly in cancer therapy. In this study, an environmentally friendly approach was developed to synthesize SeNPs using an aqueous leaf extract of Catharanthus roseus (L.), which served as both the reducing and stabilizing agent for converting H₂SeO₃ into elemental selenium nanoparticles. The extraction process was optimized at a leaf-to-water ratio of 20 g per 100 mL of double-distilled water, with boiling for 70 min. The optimal conditions for SeNP synthesis were 12 mL of leaf extract mixed with 30 mL of 5 mM H₂SeO₃ at pH 8 and 80 °C for 105 min. Morphological analysis revealed that the biosynthesized SeNPs were predominantly spherical, with an average particle size of approximately 50 nm. GC–MS and FTIR analyses demonstrated the presence of phytochemicals in the leaf extract that participated in nanoparticle formation while providing surface capping and stabilization. The biological activity of the synthesized SeNPs was evaluated against SK-LU-1, Hep-G2, and MCF-7 cancer cell lines. After 72 h of treatment, the nanoparticles exhibited potent cytotoxicity, with half-maximal inhibitory concentration (IC₅₀) values of 5.54 μg/mL, 4.45 ��g/mL, and 4.93 μg/mL, respectively. These findings indicate that Catharanthus roseus (L.)-mediated SeNPs represent a simple, sustainable, and effective nanomaterial with promising potential for anticancer applications.