Inspired by antitumor drug 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), a series of novel nitrobenzoxadiazole-piperazine hybrids were designed, synthesized, and evaluated for their anticancer activities in vitro. The representative hybrids 3u, 3v, and 3w containing a chloromethyl substitution exhibited the most potent anticancer effects against MCF-7, HepG2, and A549 cells. Among the synthesized compounds, 3u was found to be the most active derivative possessing broad-spectrum cytotoxicity against HepG2 and A549 cells with the highest IC50 values of 3.50 and 4.73 μM, respectively. Mechanistic studies of anti-proliferative activity revealed that compound 3u triggered apoptosis in cancer cell lines through hydrolyzing of PARP by caspase 3, and effectively inhibited colony formation. These results suggested that compound 3u deserves further development as a promising lead compound for discovering novel antitumor agents.