Interaction of some cyclic chalcone analogs, (E)-2-(4′-X-benzylidene)-1-benzosuberone derivatives, with calf thymus DNA has been investigated using thin-layer chromatographic and UV–Vis spectroscopic methods. Interaction of the compounds with calf thymus DNA exhibited relatively high intrinsic binding constants in the range of 3.1 × 104–1.0 × 105 M−1. The results indicate existence of weak, non-covalent interactions between the investigated chalcones with calf thymus DNA. Furthermore, the DNA cleavage activity of the compounds was studied by means of agarose gel electrophoresis. Each compound showed a slight DNA cleavage activity with pBR322. The obtained results provide additional knowledge on the previously documented cytotoxicity against several tumor cell lines of the cyclic chalcone analogs.