Two ruthenium(II) complexes [RuIICl(PPh3)2(L)] (1) and [RuII(L)2] (2) were synthesized by reacting [RuCl2(PPh3)3] and thiophene-2-carboxylic acid (1-pyridine-2-yl-ethylidene)-hydrazide (HL) in methanol–chloroform, and characterized by elemental analysis and spectral and XRD data. The ratio of ligand to metal is 1:1 in the former complex and 2:1 in the latter. Interaction of these complexes with CT-DNA was studied using absorption and emission spectral studies; these show that both the complexes interact with CT-DNA through intercalative modes of interaction. Their BSA-binding activity results indicated the operation of static quenching mechanism and stronger binding of tryptophan residues than tyrosine residues. In vitro cytotoxicity assays against HeLa and MCF-7 cell lines showed better activity of both the complexes compared to the standard drug cisplatin. Overall, the activity of complex 2 with two units of coordinated ligand showed better activity than the other one.