Synthesis of gold nanoparticles (AuNPs) using phytochemicals has become tremendously prominent in biomedical applications because of its enhanced bioactivity and biocompatibility. In this study, water extracts from the leaves of Cyclopia genistoides (C. genistoides), commonly known as honeybush (HB), were used to synthesize honeybush gold nanoparticles (HB-AuNPs). The HB water extracts (HBE) served as both reducing and capping agents in the synthesis of HB-AuNPs. The HB-AuNPs were characterized by UV–Vis spectrophotometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The cytotoxicity and apoptotic effects of the HBE and HB-AuNPs, alone and in combination with doxorubicin (Dox), were examined against various human cell lines. Spherical-shaped HB-AuNPs with a hydrodynamic diameter range of 63 to 121 nm were produced. The HB-AuNPs conferred selective cytotoxicity against colon (Caco-2), breast (MCF-7), and prostate (PC-3) cancer cells and did not display any cytotoxicity to non-cancerous skin fibroblast (KMST-6) and human embryonic kidney (HEK)-293 cells. Moreover, co-treatment of Caco-2 cells with HB-AuNPs and Dox (at non-toxic concentrations) significantly enhanced the anti-cancer effects of Dox towards the Caco-2 cells. Furthermore, it was demonstrated that HB-AuNPs induced PC-3 cell death through apoptosis. Further studies are warranted to elucidate the mechanisms by which the HB-AuNPs influence cell death when used alone or in combination with drugs, as well as the type of phytochemicals involved in AuNPs synthesis and activity.