Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) transition metal complexes of 2-hydroxy-5-[(E)-(4-phenyl) diazenyl] benzaldehyde oxime and 2-hydroxy-5-[(E)-(4-nitrophenyl) diazenyl] benzaldehyde oxime ligands were synthesized and characterized through NMR, IR, ESI mass, and UV analysis. DNA binding abilities of the complexes were revealed using a UV-Vis spectrophotometer with the absorption titration and competitive binding techniques. Hydrolytic and oxidative DNA cleavage activities under different conditions were also proved. Topoisomerase I inhibition efficiencies and in vitro free radical scavenging activities of all complexes were examined. Finally, the selective cytotoxic potentials of all complexes were evaluated in human colon cancer, normal colon, and fibroblast cell lines using the water-soluble tetrazolium salt (WST-1) assay. The complexes had the ability to intercalate into stacked base pairs of DNA and topoisomerase I activity was reasonably inhibited in their presence in 0.4 mM concentrations. The abilities for scavenging of DPPH and hydroxyl radicals were found to be higher than those of known standard antioxidants (ascorbic acid, butylated hydroxyanisole, and mannitol). The results obtained from the cytotoxicity experiments are especially promising for further research, which must be carried out for the evaluation of the studied complexes as anticancer drugs.