A series of 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazoles have been synthesized and their ability to inhibit the growth of cancer cell lines has been investigated against NCI 60 cell panel. The compound 1-[(2E)-3-phenylprop-2 enoyl]-2-(4-chlorophenyl)-1H-benzimidazole (Compound 3f, NSC: 773404/1) was found to display good antitumor activity against the nine tumor subpanels with the selectivity ratios ranging from 0.79 to 1.53 and from 0.47 to 1.69 at GI50 and TGI levels, respectively. Structural parameters of the potent anticancer active compound, 3f have been studied from single crystal XRD data. Further, in vitro interaction of 3f with transport protein, human serum albumin (HSA) was investigated by spectroscopic techniques. Static quenching mechanism was noticed in the binding of 3f to HSA. Thermodynamic parameters revealed that the hydrogen bond and van der Waals forces played a major role in the interaction process.