A series of novel functionalized 1,3-selenazole was synthesized by Hantzsch-type condensation reaction and evaluated for their in vitro cytotoxic activity against two human cancer cell lines. The structures of the synthesized selenosemicarbazones and functionalized 2-hydrazinyl-1,3-selenazole derivatives were assigned based on IR and NMR spectroscopic investigations, mass spectrometry, and elemental analysis data. Some of the synthesized compounds showed significant cytotoxic activities, the IC50 values below or around 10 lM were recorded with compounds 1d and 4b on both androgeninsensitive prostate cancer cells (DU-145) and hepatocarcinoma (Hep-G2) cell lines.