A series of novel chiral ruthenium(II) complexes with phenolic hydroxyl groups were synthesized and characterized. These ruthenium(II) complexes exhibited strong dual inhibition of topoisomerases I and IIα, with approximate IC50 values of 3-15 mM, which were more efficient than the widely clinically used single TopoI poison camptothecin (CPT) or TopoIIα poison etoposide (VP-16). Δ-1 and Λ-1 with more hydroxyls were observed to be more potent inhibitors. To further evaluate the mechanism of the complexes at a cellular level, these complexes were investigated for their effect on cell proliferation, cell cycle progression and induction of apoptosis. The results indicated that ruthenium(II) complexes permeated the nuclei in cancer cells and inhibited the activities of nuclear enzymes topoisomerases I and IIα, then triggered DNA damage and induced apoptosis in the cancer cells. The simultaneous inhibition of TopoI and TopoIIα induced the death of cancer cells, which may be a promising and effective strategy for cancer therapy.