Ruthenium complexes are currently significant attention in medicinal chemistry as they offer various properties which make them an appropriate choice for drug development. Herein, a series of ruthenium(II)-p-cymene-2-aryl-imidazo-1,10-phenanthroline derivatives have been prepared and characterised by elemental analysis, infrared, LC-mass and NMR techniques. The structural and chemical properties shows that Ru(II) complexes have got rigidity, planarity, aromaticity, hydrogen donating and accepting capability which aids both solubility and interaction with biomolecules. The binding strength of these complexes with DNA and BSA were found to be 104–106 M−1. The competitive displacement of ethidium bromide (EtBr) from DNA in the presence of complex reveals an intercalation or groove binding further this was supported by viscosity and in-silico studies. The cytotoxicity study of these Ru(II) complexes were conducted with two cancer cell lines (MDA-MB-231 and HeLa) and one human embryonic kidney cells (HEK-293). The study revealed that [(η6-p-cymene)RuCl (κ2-N,N-2-(4-fluorophenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline].PF6 (4e), [(η6-p-cymene)RuCl(κ2-N,N-2-(4-bromophenyl)-1H-imidazo[4,5-f][1,10]Phenanthroline].PF6 (4f) and [(η6-p-cymene)RuCl(κ2-N,N-2-(4-nitrophenyl)-1H-imidazo[4,5-f][1,10]Phenanthro line].PF6 (4g) were found exhibit least inhibitory concentration (IC50) and high selectivity with respect to HeLa and MDA-MB-231. The activity of the Ru(II) complexes were position and substituents dependent. © 2020 Elsevier B.V.