A series of mononuclear ruthenium arene complexes with thiosemicarbazone (TSC) ligands (A-type, 1–8) and their corresponding di-nuclear analogues (B-type, 9–16) were synthesized and characterized by NMR, elemental analysis and HR-ESI-mass spectrometry. The molecular structures of 1, 2, 6, 9–11 and 13–16 were determined using single-crystal X-ray diffraction analysis. The Gibbs free energy of the two examples of the two types of complexes (1 and 9) and the bonding order in their single-crystals were studied using density functional theory (DFT) calculations. The compounds were further evaluated for their in vitro antiproliferative activities against CNE-2 human nasopharyngeal carcinoma, KB human oral epithelial carcinoma, SGC-7901 human gastric carcinoma, HepG2 human liver carcinoma, HeLa human cervical carcinoma and HEK-293T noncancerous cell lines. Furthermore, the interactions between the compounds and DNA were studied by electrophoretic mobility spectrometry studies.