This contribution insight on the cytotoxic and anticancer activities and molecular mechanism of phyto-reduced silver nanoparticles (AgNPs) in MCF-7 breast cancer cell lines. A simple, entirely green synthesis process was optimized for the phyto-reduction of AgNP (~12.7 nm) using aqueous leaf extracts of Indigofera heterantha. The structural and vibrational properties of biosynthesized AgNPs were extensively characterized using UV-Vis spectrophotometer, x-ray diffraction (XRD), dynamic light scattering (DLS), and Fourier transform Infrared spectroscopy (FTIR), while their shape and morphology was studied through scanning electron microscopy (SEM). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay indicates concentration dependent inhibition with IC, 27.93 ± 2.10 μg/mL against MCF-7 cells and 294.38 ± 3.87 μg/mL against L929 cells. The manifested anticancer mechanism in MCF-7 cells was extensively studied using Acridine orange/ethidium bromide (AO/EB), 4',6-diamidino-2-phenylindole (DAPI) and Annexin-V/propedium iodide fluorescence microscopic assays. The level of reactive oxygen species (ROS) was measured using DCFH-DA fluorescent spectroscopy. Overall, the results show that AgNPs exhibit cytotoxic and apoptotic effect on breast cancer MCF-7 cells by damaging membrane integrity and nuclear fragmentation due to oxidative stress generated by elevated level of ROS. RESEARCH HIGHLIGHTS: Biomimetic synthesis of nano dimension size silver nanoparticles (AgNPs). Characterization of AgNPs through UV-Vis, DLS, XRD, FTIR, and SEM. Cytotoxic and anticancer effects of the biosynthesized AgNPs in L929 fibroblast cells and MCF7 breast cancer respectively. Determination of morphological, and nuclear changes triggered by AgNPs in MCF 7 breast cancer cells using fluorescent microscopy and flow cytometry. Apoptosis induction by AgNPs in cancer cells through oxidative stress generated by reactive oxygen species (ROS).