Green synthesis is a new paradigm for the preparation of gold nanoparticles (AuNPs) due to its cost-effectiveness and favorable environmental impact. This study presented a simple phytosynthesis process for the preparation of AuNPs utilizing the aqueous peel extract of Spondias dulcis (SPE) (Anacardiaceae) as both a reducing and stabilizing agent. A visual color change from yellow to purple during the reaction implied the successful formation of SPE-AuNPs, which was confirmed by UV–vis spectroscopy. Transmission electron microscopy (TEM) images indicated that the SPE-AuNPs were predominantly spherical with a mean size of 36.75 ± 11.36 nm, and were comprised of crystalline Au, as indicated by X-ray diffraction. In terms of their potential application, SPE-AuNPs exhibit significant cytotoxic activity in a dose- and time-dependent manner to MCF-7 human breast cancer cells, while being non-toxic to Vero normal cells. The treatment of MCF-7 cells with SPE-AuNPs increased the production of intracellular reactive oxygen species (ROS). Herein, the findings highlight the potential contribution of phytosynthesized SPE-AuNPs to the development of novel nanomedicines for cancer treatment.