This study reports the synthesis, characterization, and biological evaluation of four novel heteronuclear Ru(II)-Cu(II) complexes with phosphine-fluoroquinolone conjugates. Structural analysis using X-ray diffraction, density functional theory calculations, and various spectroscopic techniques confirmed the stability and coordination geometries. The cytotoxicity was evaluated in vitro across multiple cancer cell lines (lung, breast, pancreatic, prostate) and noncancerous cells. In vivo toxicity was also assessed using a zebrafish () larvae model. The results demonstrated that Ru(II)-Cu(II) complexes exhibit greater anticancer activity compared with cisplatin while almost being nontoxic toward control cells in vitro. Mechanistic studies revealed that their action involves nuclei accumulation, reactive oxygen species generation, lipid peroxidation, and mitochondrial membrane depolarization. The expression ratio of Bax to Bcl-2 mRNA correlates with apoptotic cell death. Additionally, encapsulation of the one Ru(II)-Cu(II) complex in bilosomes significantly improved its stability, selectivity, and therapeutic efficacy in three-dimensional (3D) cancer spheroid models.