Five sesquiterpene lactones were isolated and identified from Ambrosia maritima L.. Hymenin showed highest cytotoxic activity against HCT-116, A-549 and MCF-7 cell lines (IC 50= 3.83 ± 0.2, 5.48 ± 0.3, 10.1 ±0.6 µg/ml respectively). Damsin has significant COX-2 inhibitory activity (IC 50 33.97 ± 1.62 µg/ml) while hymenin showed highest selectivity to COX-1 (IC 50 = 18.21 µg/ml) and significant inhibition of NO (IC 50 18.19 ± 0.75 µg/ml). From docking study, maritimolide showed higher affinity towards human Src kinase than the native ligand, Bosutinib. Results suggested that both COXs/ Src kinase inhibition could contribute even partially to the overall mechanism of cytotoxic activity of the five compounds. The structure activity relationship revealed that α-methylene-γ-lactone moiety enhances the activity against tested cell lines, OH group at C-1 increase activity of hymenin. However; the reduction of the double bond at C-2 as in damsin resulted in significant decrease in activity against HCT-116 and MCF-7 cells.