Chromomycin A3 (Chro) has been evidenced to exhibit much higher binding affinity toward Fe(II) by forming a highly stable 2:1 drug/metal complex, compared to its structural analogue, mithramycin (Mith). Different properties of the [(Chro)2-Fe(II)] complex acting on DNA, such as sequence specificity, DNA cleavage, and topoisomerase I (TopI) inhibition were studied. Kinetic analyses of surface plasmon resonance showed that the affinity of the [(Chro)2-Fe(II)] complex upon binding to hairpin DNA duplexes containing various tetranucleotide sequences follows the order: GGCC > CGCG > CCGG approximately GCGC > AGCT > ACGT > TGCA > TCGA. According to circular dichroism (CD) studies, most hairpin DNA duplexes appeared to retain their B-type conformations in the presence of the [(Chro)2-Fe(II)] complex, except the duplex containing the GGCC sequence, which exhibited the features of both A- and B-type DNA. In DNA-cleavage assays, the [(Chro) 2-Fe(II)] complex was shown to cause single-stranded cleavage of plasmid DNA because of a Fenton-type reaction. DNA cleavage activity of the [(Chro) 2-Fe(II)] complex was increased at low pH. Moreover, the complex was capable of inhibiting TopI activity. The [(Chro)2-Fe(II)] complex exhibited higher cytotoxicity than the [(Mith) 2-Fe(II)] complex in several cancer cell lines, most likely owing to its more stable dimeric structure and higher DNA-binding affinity. Our results provide significant evidence that the [(Chro)2-Fe(II)] complex could be promising in terms of its biological applications in the future.