This study showed that bio-functional silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) were synthesized in aqueous extracts of Gymnema sylvestre leaves and tested for toxicity assessment against triple-negative breast cancer cells (TNBC). Biofunctional nanoparticle (NPs) samples were characterized using UV-Vis spectroscopy, FT-IR, XRD, SEM, and TEM. The results showed that the phytofabrication of AgNPs resulted in a dark brown, UV-vis maximum absorbance peak at 413 nm. The AgNPs were crystalline and spherical, with sizes ranging from 20 to 60 nm, as confirmed by the XRD pattern and TEM images. Another phytofabrication of ZnONPs exhibited a white precipitate corresponding to a UV-Vis maximum absorption peak at 377 nm and a fine micro flower morphology with a particle-sized tribution between 100 and 200 nm. In addition, FT-IR spectra showed that bioorganic compounds are associated with NPs that respond to reduced Ag+ ions and AgNPs tabilizers. Invitro cytotoxicity studies revealed the potent anti-cancer effects of phytofabricated AgNPs and ZnONPs on TNBC cells. Furthermore, the AO/EB double staining assay results proved that apoptotic cells are distinguished by greenish-yellow fluorescence of the cell nuclei with IC50 concentrations of 44 ± 0.8 µg/mL for AgNPs and 26.2 ± 0.5 µg/mL for ZnONPs, respectively. Based on our results, we expect that the anticancer function of the biofunctional NPs is due to the apoptotic activation of TNBC cells by increased ROS. Therefore, the presented study demonstrated that biofunctional AgNPs and ZnONPs have excellent prospects for the anti-cancer activity that can be used in pharmaceutical and medical fields.