In search of potential biologically active compounds, some novel 2,5-disubstituted 1,3,4-oxadiazole derivatives have been prepared conveniently via oxidation of newly synthesized pyrazolylaldehyde N-isonicotinoyl hydrazones by (diacetoxyiodo)benzene in dichloromethane under mild reaction conditions. Compounds were obtained in excellent yields, and their structures have been established on the basis of their FT-IR, 1H, 13C NMR, and mass spectral data. The DNA photocleavage potential for all the synthesized compounds was evaluated using agarose gel electrophoresis. It has been observed that oxadiazole derivatives showed a significant level of DNA photocleavage activity when compared with their corresponding hydrazones, and some modifications in the basic structure may lead to construct some potential chemotherapeutic agents in future.