A series of thirty two novel pyrrolo[2,3-b]pyridine analogues synthesized, characterized ((1)H NMR, (13)C NMR and MS) and cytotoxic evaluation of these molecules carried out over a panel of three human cancer cell lines including A549 (lung cancer), HeLa (cervical cancer) and MDA MB-231 (breast cancer), using sulforhodamine B assay method. Few molecules such as 5c, 5d, 5e, 5h, 5k, 5m, 5n, 5q, 5r, 7f, 7j, 7g and 7k exhibited maximum growth inhibitory action against the tested cancer cell lines at lower micro molar concentration. Noticeably, compounds exhibited good growth inhibition in all three cancer cell lines in the range of 0.12 μM-9.84 μM. Further study exposed that one of the active compound 5d could efficiently intercalate into calf thymus DNA to form 5d-DNA complex which might block DNA replication to influence their antiproliferative activity. The molecular interactions of all the synthesized analogs were also supported by molecular docking simulations. We believe that further optimization of these compounds will lead to potential anticancer agents.