Tanacetum sinaicum, family Asteraceae is a wild plant indigenous to Egypt and grows on the rocky slopes of Sinai. Because of the large variation observed between the previous reports concerning the essential oil constituents. The present work aimed to perform a detailed investigation of the chemical composition of essential oil from T. sinaicum. Furthermore, the present study represents the first report about cytotoxic and antioxidant activities of essential oil of this plant. The essential oil was subjected to GC-MS analysis which resulted in identification of 41 constituents representing 85.81% of its total composition. The biological activity of the oil showed moderate cytotoxic activity against human hepatocellular carcinoma, colon carcinoma and human lung cancer cell lines. In addition, the antioxidant activity was investigated using the DPPH free radical assay and the results indicated that the oil has strong antioxidant activity. Furthermore, the insecticidal activity on culex pipiens mosquitoes was measured using the immersion method, the oil showed high mortality percentages for the larval, pupal and adult stages. The antimicrobial results indicate that the oil has a positive activity against the Gram negative bacteria Proteus vulgaris and Gram positive bacteria Bacillus subtilis. Also, it has some activity against Escherichia coli and Staphylococcus aureus.