Mixed-ligand copper(II) complexes with 4,5-dichloro-isothiazole-3-carboxylic acid and heterocyclic N-donor ligands as potential anticancer agents. Display omitted •Mixed-ligand Cu(II) polypyridine complexes with isothiazole derivative were prepared.•SC-XRD revealed square bipyramid and square pyramidal geometry of complexes.•Formation of Cu(L)(polypyridine)(H2O)+ clusters was revealed in aqueous solution.•Dose-dependent cytotoxicity of 3–5 was shown against Hep-2 and MCF-7 tumor cells.•Cu(dmphen)L2 possessed highest cytotoxicity on Hep-2 cells (IC50 = 0.97 ± 0.03 µM). Mixed-ligand copper(II) complexes based on 1,10-phenanthroline and related compounds are of interest to scientists due to their promising anticancer properties. In this study, four new water-soluble copper(II) complexes Cu(dmbipy)L2, Cu(phen)(H2O)L2, Cu(dmphen)L2 and Cu2(bipy)2L4, where HL – 4,5-dichloro-isothiazole-3-carboxylic acid, bipy – 2,2′-bipyridine, dmbipy – 2,2′-bi-4-picoline, phen – 1,10-phenanthroline, dmphen – 4,7-dimethyl-1,10-phenanthroline are reported. All complexes have been characterized by elemental and powder X-ray diffraction analysis, EPR and IR-spectroscopy. Molecular structures of the reported complexes have been determined by single crystal X–ray diffraction. Copper(II) ion, HL and heterocyclic N-donor ligands have been found to form 1:2:1 complexes that possess square bipyramid or square pyramidal geometry. The UV–vis spectroscopy and mass spectrometry have been applied to show the behavior of the compounds in solution. All complexes have been screened in vitro for their cytotoxic activity against Hep-2 and MCF-7 cell lines. They exhibit significant dose-dependent cytotoxic effect and Cu(dmphen)L2 is found to be the most cytotoxic (IC50 = 0.97 ± 0.03 µM when compared to IC50 = 9.2 ± 0.5 µM for control, cisplatin – Hep-2 cell line). The investigation of DNA binding ability by UV–vis titration technique indicates that complexes obtained exhibit moderate binding affinity toward calf thymus DNA. Effect of Cu(dmbipy)L2 and Cu(dmphen)L2 on activity of drug-metabolizing enzymes cytochromes P450 has also been investigated. The addition of complexes to the hepatic microsomes of 3-MC or PB-treated rat, lead to a dose-dependent decrease of CYP’s activities. The data obtained indicate that Cu(dmphen)L2 and Cu(phen)(H2O)L2 can be potential anticancer agents.