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In the present investigation, we report the synthesis and characterization of three novel nickel(II) [Ni(L)2] (1) and copper(II) [Cu(L)2] (2a) and [Cu(L)2] (2b) complexes, which are made through the coordination of (E)-1-(2-methoxybenzylidine)-4-phenylethiosemicarbazone (HL) as bidentate N,S-ligand. Several modern techniques including experimental electronic and fluorescence spectroscopy, single-crystal X-ray crystallography, molecular docking and BSA-binding are used to characterize the isolated coordination compounds. X-ray crystallography, FT-IR and UV–visible spectra agree with the observed crystal structures. The crystallographic and spectroscopic studies confirmed that these complexes display four-coordinate square-planar trans-[MN2S2] coordination geometry, whose central metal(II) atom lies on the center of symmetry. Complexes 1 and 2a crystallized in the orthorhombic system of the space group Pbca whereas 2b crystallized in the monoclinic system of P21/c. The binding affinity of complexes towards bovine serum albumin (BSA) was determined by UV–visible and fluorescence spectrophotometric methods. The cytotoxic/antiproliferative potential of the synthesized compounds on human cell lines was also investigated by MTT assay. [ABSTRACT FROM AUTHOR]