In this communication, two new Ru(II)-arene-ninhydrin complexes [(η6-p-cymene)RuCl(k2-N,O-2-(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)hydrazine-1-carboxamide (Ru-1) and [(η6-p-cymene)RuCl(k2-N,S-2-(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)hydrazine-1-carbothioamide (Ru-2) have been prepared and analyzed by different analytical and spectroscopic techniques. The studies show that Ru(II) complexes have “piano stool” coordination geometry, comprises of one π-bonded arene centroid, two σ-bonded nitrogen atoms and one chlorine atom from ninhydrin thiosemicarbazole and Ru(II) metal centre respectively. The molar conductivity study of these complexes in DMSO:water (9:1, v/v) media reveals that complexes are ionic in nature. The photo-physical properties of Ru-1 and Ru-2 with calf thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA) were explored. The intrinsic binding constant (Kb) of Ru-1 and Ru-2 with DNA/BSA was found to be 4.5 × 105 M−1/1.3 × 105 M−1 and 2.1 × 105 M−1/1.2 × 105 M−1 respectively. The competitive displacement of ethidium bromide (EtBr) from DNA in the presence of Ru-1 and Ru-2 quantified by quenching constant and viscosity studies. This high binding ability is due to groove binding or intercalation of Ru(II) complexes with CT-DNA which is well supported with in-silico studies. The effect of the Ru-1 and Ru-2 were examined on two different cancer cell lines (HeLa and MDA-MB-231) using the MTT assay. The obtained results show that Ru-1 and Ru-2 exhibits high potency and selectivity with HeLa (IC50 = 9.5 × 10−6 mol/L) and MDA-MB-231(IC50 = 14.7 × 10−6 mol/L) cell lines respectively. © 2020