This study synthesized gold nanoparticles (AuNPs) using a facile microwave-assisted chemical route and evaluated them as potential anticancer candidates against breast and colon cancer cell lines. Numerous spectral characterization tools were used to study the optical properties, structure, and morphology of the prepared AuNPs. UV-Vis spectroscopy showed a characteristic peak at 517 nm, which confirms the formation of AuNPs. The crystalline structure of NPs was studied by X-ray diffraction, and the NPs’ shape and size were calculated with Field emission transmission electron microscopy. The synthesized AuNPs were found to be uniform in size in the range of 2–6 nm. A variety of biological tests, including MTT, scratch, real time polymerase chain reaction (RT-PCR), and comet assays were adopted to assess the anticancer potential of these AuNPs in the studied cancer cell models. The findings suggested a cell-dependent cytotoxicity of AuNPs. Different cell viability of 40.3 and 66.4% were obtained for MCF-7 and HCT-116, respectively, at 5 µg/mL of AuNPs. The scratch assay showed AuNPs impede cell migration in a concentration-dependent manner in the MCF-7 cell line. On the other hand, real-time polymerase chain reaction (RT-PCR) of apoptotic (p53, Bax, and caspase-3) and anti-apoptotic (BCl-2) genes revealed upregulation and downregulation of these genes, respectively, probably leading to its cytotoxicity. At 5 µg/mL concentration of AuNPs, reactive oxygen species (ROS) production was found to be increased by 26.4 and 42.7%, respectively, in MCF-7 and HCT-116 cells. Similarly, comet assay demonstrated AuNPs induced DNA damage in the studied cancer cell lines. These findings suggest that the observed anticancer efficacy of AuNPs was driven by ROS generation. The synthesized AuNPs appeared to be a promising therapeutic against cancer cells. However, our data need to be confirmed and validated in and models so that this NP can be further exploited for human use.