Phytochemical mediated synthesis of nanoparticles has gained great interest in the field of cancer therapeutics. We attempted a simple and stable synthesis of gold nanoparticles (AuNPs) with Myricetin (Myr) adopting ultrasound-assisted method. Further, we evaluated anticancer activity of the synthesized nanoparticles. The physico-chemical properties of biosynthesized Myr-AuNPs were characterized by UV-visible spectrophotometer, Fourier-transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and particle size analysis. The study reports of Myr-AuNPs showed spherical-shaped particles with a size of < 50 nm. Stability of the particles was increased in various physiological media. Furthermore, the graph theoretical network analysis of Myr-AuNPs indicated that the probable binding with the mTOR is an effective target for breast cancer cells. In silico molecular docking study of Myr-AuNPs in human mTOR kinase was found to be strong binding. The IC50 value of Myr-AuNPs was calculated as 13 μg mL−1 against MCF-7 cell line. The AO/EB and DAPI stainings confirmed the anticancer activity by Myr-AuNPs-treated cells showed a good proportion of dead cells evidenced with formation of pro-apoptotic bodies. In addition, Myr-AuNPs exhibited depolarization of mitochondrial membrane potential and production of reactive oxygen species. This study proves that Myr-AuNPs holds great promise to use against breast cancer as a potent anticancer drug. [Figure not available: see fulltext.]. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature.