A study concerning on in-vitro anticancer evaluation of structurally tuned 1,10-phenanthroline at 2,9 positions with different functional groups such as –CH3 (S1), >C[dbnd]O (S2), –COOH (S3), –COOCH3 (S4) and –CONHNH2 (S5) were described. The solubility data revealed that all ligands were completely soluble in dimethyl sulphoxide (DMSO) and moderately soluble in water. The photo-physical properties of these ligands revealed that a common absorption peak appeared in the region of 270–300 nm and emission spectra in the region of 330–510 nm with a large Stokes shift of 85 nm. The binding constant of ligands (S1-S5) with calf-thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA) were found to be 105 M−1 and 104 M−1 respectively. The fluorescence quenching of ethidium bromide (EtBr) from DNA upon addition of ligand was confirmed from binding affinity values KSV (104 M−1) and Kapp (106 M−1). The mode of interaction of ligand with DNA is either by intercalation or groove binding this is further supported by viscosity and in-silico studies. The gel electrophoresis studies exhibited that S4 and S5 have cleaved plasmid DNA completely within 60 min while rest of the ligands took more than 60 min. The cytotoxicity study of these ligands (S1-S5) were conducted with two different cancer cell lines (MDA-MB-231 and HeLa) and their performance were compared with normal HEK-293 cells. The study revealed that ligands S5 and S4 were found to be least inhibitory concentration (IC50) and high selectivity factor values of 7.66 µM/12.97 and 13.35 µM/6.19 with respect to HeLa and MDA-MB-231 cell lines while ligands S1-S3 showed high IC50 values compare to doxorubicin. However, both S5 and S4 ligands have been displayed higher cytotoxicity effect than doxorubicin and least effect on normal cell HEK-293. © 2020 Elsevier B.V.