The green chemistry approach using plants involves phytoconstituent mediated reduction process for synthesis of nanoparticles. Biogenic Silver nanoparticles (AgNPs) were synthesized using aqueous stem extract of Ruta graveolens. The adopted biological method is compatible with green chemistry principles because the plant extract acts as a dual functional molecule: as reductant and a stabilizing agent for the synthesis of AgNPs. The UV–visible spectra confirmed that reduction of silver ions for Ruta stem extract at 485 nm on 24-hour incubation. FT-IR spectroscopic spectrum showed the peaks at 3507, 2829, 1579, 1010, 755 which are characteristic of flavonoids and terpenoids present in the stem. The zeta potential of Ruta AgNPs was found to be -35.4 mV which indicate that the nanoparticles are quite stable due to charge accumulation on its surface. The TEM micrograph reveals that the particles were of spherical shape & monodispersed, the average size of the particles is around 15 nm. Ruta AgNPs showed cytotoxicity against cancer cells and the results indicated that with the increase in Ruta AgNPs concentration there was a significant (p < 0.05) decrease in cell viability of HepG2 as compared to control. The antioxidant activity of Ruta AgNP was confirmed by ABTS radical scavenging activity. Ruta AgNPs may provide novel resources for the evaluation and development of newer, safer, and effective antimicrobial and antiproliferative drug formulations.