Four novel phosphanegold(I) complexes of the type [Au(PR3)(DMT)].PF6 (1–4) were synthesized from 3-Thiosemicarbano-butan-2-one oxime ligand (TBO) and precursors [Au(PR3)Cl], (where R = methyl (1), ethyl (2), tert-butyl (3), and phenyl (4)). The resulting complexes were characterized by elemental analyses and melting point as well as various spectroscopic techniques, including FTIR and (1H, 13C, and 31P) NMR spectroscopy. The spectroscopic data confirmed the coordination of TBO ligands to phosphanegold(I) moiety. The solution chemistry of complexes 1–4 indicated their stability in both dimethyl sulfoxide (DMSO) and a mixture of EtOH:H2O (1:1). In vitro cytotoxicity of the complexes was evaluated relative to cisplatin using an MTT assay against three different cancer cell lines: HCT116 (human colon cancer), MDA-MB-231 (human breast cancer), and B16 (murine skin cancer). Complexes 2, 3, and 4 exhibited significant cytotoxic effects against all tested cancer cell lines and showed significantly higher activity than cisplatin. To elucidate the mechanism underlying the cytotoxic effects of the phosphanegold(I) TBO complexes, various assays were employed, including mitochondrial membrane potential, ROS production, and gene expression analyses. The data obtained suggest that complex 2 exerts potent anticancer activity against breast cancer cells (MDA-MB-231) through the induction of oxidative stress, mitochondrial dysfunction, and apoptosis. Gene expression analyses showed an increase in the activity of the proapoptotic gene caspase-3 and a reduction in the activity of the antiapoptotic gene BCL-xL, which supported the findings that apoptosis had occurred.