Ru^II(η⁶-arene) complexes, especially with bioactive ligands, are considered to be very promising compounds for anticancer drug design. We have shown recently that Ru^II(η⁶-p-cymene) complexes with 3-hydroxyflavone ligands exhibit very high in vitro cytotoxic activities correlating with a strong inhibition of topoisomerase IIα. In order to expand our knowledge about the structure–activity relationships and to determine the impact of lipophilicity of the arene ligand and of the hydrolysis rate on anticancer activity, a series of novel 3-hydroxyflavone derived Ru^II(η⁶-arene) complexes were synthesised. Furthermore, the impact of the heteroatom in the bioactive ligand backbone was studied by comparing the cytotoxic activity of Ru^II(η⁶-p-cymene) complexes of 3-hydroxyquinolinone ligands with that of their 3-hydroxyflavone analogues. To better understand the behaviour of these Ru^II complexes in aqueous solution, the stability constants and pK_a values for complexes and the corresponding ligands were determined. Furthermore, the interaction with the DNA model 5′-GMP and with a series of amino acids was studied in order to identify potential biological target structures.