Tanacetum sinaicum (Asteraceae) is a rare herb growing wild in Southern Sinai mountains in Egypt. The aim of this work was to develop a comparative study between the chemical compositions of the wild plant and the micropropagated one, using HPLC− ESI− MS/MS technique. HPLC- ESI-MS/MS analysis resulted in the identification of 56 compounds including flavonoids, organic acids and their glycosides. Totally, 52 compounds were tentatively identified from the methanolic extracts of wild plant, 10 of them could also be detected in the methanolic extract of the micropropagated plant, in addition to 4 compounds that were detected only in the micropropagated plant. Methanolic extracts of the wild and micropropagated plants showed moderate cytotoxic activity against Hep-G2, HCT-116 and A-549 cell lines using MTT cell viability assay. The antioxidant activity using the DPPH free radical assay was investigated and the results indicated that the wild plant methanolic extract has better antioxidant activity compared to the in vitro plant extract. In addition, the insecticidal activity was measured using the immersion method and the methanolic extract of the micropropagated plant showed a higher mortality percentage than wild plant extract on culex pipiens. Also, results indicate that wild plant extract has mild antimicrobial activity against Gram positive bacteria Staphylococcus aureus and Bacillus subtilis and showed no activity on both fungi and Gram negative bacteria. while in vitro plant extract showed some activity against Gram positive bacteria Staphylococcus aureus and the Gram negative bacteria Escherichia coli.