A series of simple quinoline–chalcone conjugates have been synthesized by Claisen–Schmidt condensation reactions of substituted acetophenones with 2-chloro-3-formyl-quinoline and evaluated for their nucleolytic activity. The structures of the synthesized quinoline–chalcone conjugates were confirmed by IR, ¹H NMR, ¹³C NMR and mass spectral analyses. Most of the prepared compounds showed significant DNA binding and photocleavage activities. The incorporation of an electron-donating group into ring A caused a moderate increase in the DNA binding and photocleavage activities. Compounds 3c and 3d exhibited promising DNA photocleavage against pUC 19 DNA with 85% inhibition at 100 μM concentration. A structure–activity relationship analysis of these compounds was performed; compounds 3c and 3d are potential candidates for future drug discovery and development.