A string of four new hetero binuclear Ru(III) complexes of ferrocenecarboxaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized by various spectral (infrared, ultraviolet-visible, Electron Paramagnetic Resonance (EPR) and High Resolution Mass Spectrometry (HR-MS) techniques. The binding abilities of the ligands/complexes with nucleic acid (calf thymus DNA, CT-DNA) and bovine serum albumin (BSA) were analyzed by absorption and emission titration methods. The complexes exhibited better DNA binding affinity than their parent ligands. The interaction with CT-DNA was found to be intercalative and with BSA static quenching mechanism was observed. All the synthesized Ru(III) complexes were subjected to study their in vitro cytotoxicity against MCF-7 (human breast cancer) and HT-29 (human colon cancer) cell lines. Among the four complexes, complex 3 [RuCp (FF-etsc)PPh 3 ]Cl exhibited the highest cytotoxicity in MCF-7 cells and complex 4 [RuCp (FF-ptsc)PPh 3 ]Cl was the most active on HT-29 cells. K E Y W O R D S acridine orange-ethidium bromide (AO-EB)/4, 6-diamidino-2-phenylindole (DAPI) staining, morphological studies, MTT assay, Ru(III) complexes