Chemical-ecological studies of sponges show that the secondary metabolites are only defense system. Nowadays natural products with biological activities are used as drugs for treatment of human diseases. One of the most important biological activities of sponges is cytotoxic activity. This study investigated the cytotoxic activities of α-Santonin compound which extracted from Dysidea avara. In this study, dried powder of sea sponge was extracted by acetone solvent. Then, in order to isolate the α-Santonin compound, the extract was washed by silica gel column chromatography with N-hexane and ethyl acetate solvents. The IC₅₀ value of this compound was measured by XTT assay on cancer cell lines, Jurkat/E6-1, KB/C152 and healthy Hek293 cells. The α-Santonin compound with the chemical formula C₁₅H₁₈O₃, which belongs to the Sesquiterpene-lactone group, was identified by gas chromatography, with a purity of 91% in fraction No. 24. The IC₅₀ value of the α-Santonin extracted from sea sponge compared to the oral epithelial cancer cell line and lymphocyte cell line was 88.90 μg/ml and 38.09 μg/ml respectively. Our study demonstrated that α-Santonin extracted from D. avara has an extremely potent cytotoxic effect that could be used as an anticancer agent.