The present study aims to synthesize and characterize biogenic silver nanoparticles (AgNPs) from aqueous extract of Rudraksha (Eleaocarpus ganitrus) bead and to explore its efficacy for in vitro anticancer activity. Biogenic AgNPs were synthesized at three time points and characterized primarily with UV-Visible spectroscopy, FESEM and TEM techniques. The selected AgNPs were further confirmed with XRD, EDX and FTIR methods. The green synthesized AgNPs of approximately 57 nm obtained at 48 h time period were selected for the in vitro cytotoxicity assay in three cancer cell lines, PC3, U87MG and LLC1. The green synthesized AgNPs showed maximum cytotoxicity in LLC1 with an IC50 9.7 ± 1.0 μg/mL. The outcomes propose the promising candidature of synthesized AgNPs for anti-cancer efficacy.