Objective: To evaluate the in vitro cytotoxic activity of the fractionated extract as well as isolated compounds of Arum palaestinum Boiss. ( A. palaestinum) (black calla lily), and to identify the volatile components which may be responsible for the potential antitumor activity. Methods: A. palaestinum was collected from its natural habitats and subjected to phytochemical analysis for separation of pure compounds. In vitro cytotoxic activity was investigated against four human carcinoma cell lines Hep2, HeLa, HepG2 and MCF7 for the fractionated extract and isolated compounds. While, the diethyl ether fraction was subjected to GC-MS analysis as it exhibited the most potent cytotoxic effect to evaluate the active constituents responsible for the cytotoxic activities. Results: Four flavonoid compounds were isolated (luteolin, chrysoeriol, isoorientin, isovitexin) from the diethyl ether and ethyl acetate. The extracts and the pure isolated compounds showed a significant high antiproliferative activity against all investigated cell lines. The GC-MS analysis revealed the separation and identification of 15 compounds representing 95.01% of the extract and belonging to different groups of chemical compounds. Conclusions: The present study is considered to be the first report on the cytotoxic activities carried out on different selected fractions and pure compounds of A. palaestinum to provide evidences for its strong antitumor activities. In addition, chrysoeriol and isovitexin compounds were isolated for the first time from the studied taxa.