Four new cyclopentadienylruthenium(II)-acetophenone-4(N)-substituted thiosemi-carbazone complexes, with the general formula [Ru(ƞ5-C5H5)(H-Aptsc)PPh3].Cl (1), [Ru(ƞ5-C5H5)(H-Apmtsc)PPh3].Cl (2), [Ru(ƞ5-C5H5)(H-Ap-etsc)PPh3].Cl (3) and [Ru(ƞ5-C5H5)(H-Ap-ptsc)PPh3].Cl (4) were synthesised and characterised (1H NMR, 13C NMR, IR and electronic spectroscopy). The molecular structure of representative complexes 2 and 3 was confirmed by single crystal X-Ray diffraction technique. The binding ability of complexes (1–4) to calf-thymus DNA (CT DNA) and Bovine Serum Albumin (BSA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic and an intercalative binding mode have been proposed for the complexes with CT-DNA. The BSA protein binding studies have been monitored by quenching of tryptophan and tyrosine residues in the presence of complexes and static type of quenching mechanism has been proposed. In vitro free radical scavenging activity was performed by DPPH radical. The complexes (1–4) exhibited highest scavenging activity than conventional standard vitamin C (IC50 = 5.65 ± 0.12). Further, antibacterial activity of the complexes has been screened against four pathogenic bacteria such as Salmonella paratyphi, Staphylococcus aureus, Escherichia coli and Bacillus subtilis. From the results it is found that all the complexes exhibited significant activity against the pathogens and among them, complex 3 exhibited higher activity.