Herein we present four new ruthenium(II) complexes: [Ru(mtz) 2 (dppb)] (1), [Ru(mmi) 2 (dppb)] (2), [Ru(dmp) 2 (dppb)] (3), and [Ru(mpca) 2 (dppb)] (4), where mtz = 2-mercaptothiazoline; mmi = 2-mercapto-1-methyl-imidazole; dmp = 4,6-diamino-2 -m e r c a p t o p y r i m i d i n e ; m p c a = 6 -m e r c a p t o p y r i d i n e -3 -c a r b o x y l i c a c i d ; dppb = 1,4-bis(diphenylphosphino)butane. In vitro cell culture experiments revealed cytotoxic activity for complexes 2, 3 and 4 against MCF-7 (breast, non-invasive), MDA-MB-231 (breast, invasive), A549 (lung), DU-145 (prostate) and HepG2 (liver) tumor cells, in some cases lower than the half maximal inhibitory concentration (IC 50 ) for the reference drug (cisplatin). The deoxyribonucleic acid (DNA) interactions studied by viscosity measurements, gel electrophoresis and square-wave voltammetry indicated that the DNA binding affinity primarily occurs through non-covalent interactions. Only complex 2 was able to fully inhibit the DNA supercoiled relaxation mediated by human topoisomerase IB (Top IB). The analysis indicates that complex 2 inhibits the cleavage and the reconnection steps of the catalytic cycle, being both a poison and a catalytic inhibitor.