The synthesis, structural characterization and biological activity of eight ortho-quinone(N-aryl)-oximine rhenium(i) complexes are described. The reaction of the halogenido complexes (CO)5ReX (X=Cl (4), Br (5)) with 2-nitroso-N-arylanilines {(C6H3ClNO)NH(C6H4R)} (R = p-Cl, p-Me, o-Cl, H) (3a–d) in tetrahydrofurane (THF) yields the complexes fac-(CO)3XRe{(C6H3ClNO)NH(C6H4R)} (6a–d, 7a–d) with the tautomerized ligand acting as a N,N′-chelate. The substitution of two carbonyl ligands leads to the formation of a nearly planar 5-membered metallacycle. During coordination the amino-proton is shifted to the oxygen of the nitroso group which can be observed in solution for 6 and 7 by 1H NMR spectroscopy and in solid state by crystal structure analysis. After purification, all compounds have been fully characterized by their 1H and 13C NMR, IR, UV/visible (UV/Vis) and mass spectra. The X-ray structure analyses revealed a distorted octahedral coordination of the CO, X and N,N′-chelating ligands for all Re(i) complexes. Biological activity of four oximine rhenium(i) complexes was assessed in vitro in two highly aggressive cancer cell lines: human metastatic melanoma A375 and human chronic myelogenous leukemia K562. Chlorido complexes (6a and 6c) were more efficient than bromido compounds (7d and 7b) in inducing apoptotic cell death of both types of cancer cells. Melanoma cells were more susceptible to tested rhenium(i) complexes than leukemia cells. None of the ligands (3a–d) showed any significant anticancer activity.