This work includes the synthesis of some BODIPY-derived heterocyclic Calix[4]arene compounds and the investigation of their effects on DNA. Among the calixarene-BODIPY-based target compounds, compounds 3 and 9 were newly synthesized, and compound 7 was synthesized as given in the literature. The structures of all synthesized compounds were determined by elemental analysis and spectroscopic techniques (13C NMR, 1H NMR, FT-IR). The effects of the synthesized Calixarene/BODIPY compounds (3, 7, and 9) against ct-DNA were examined via UV–Vis spectrometric and fluorimetric methods. The obtained results indicated that compounds 3, 7, and 9 bind to DNA, and the calculated binding constants (Kb) were found 1.62 × 103 M−1, 1.54 × 103 M−1, and 1.14 × 102 M−1 respectively. Also, the DNA cleavage activity was examined against pBR322 plasmid DNA. The results showed that all compounds (3, 7, and 9) had a quite high effect on pBR322 plasmid DNA. Molecular docking studies were also performed to gain insight into the binding affinities and key interactions of DNA-synthesized compounds. The strong binding of compounds to the DNA can be attributed to high negative values of binding free energy between -20.2 and -17.4 kcal mol−1. These results gave the idea that these molecules are candidate molecules, especially for anti-bacterial and anticancer studies.