Background: Phoenix dactylifera (Palmacea), known as date palm is a widespread economical plant in the Middle Eastern. The dietary fiber in P. dactylifera seeds has important therapeutic use in medical condition such as diabetes, obesity, hypertension, colorectal, and prostate cancers. Objectives: The objective is to isolate, characterize the major bioactive components and evaluate the cytotoxic activity of extract and isolated pure compound of P. dactylifera. Materials and Methods: P. dactylifera extract (DE) was obtained by maceration. The pure compound, identified as oleic acid (OA) was isolated by column chromatography. Cytotoxicity assessment was done by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay and morphological alterations in HepG2, A-549, and MCF-7 cells and bioactive compounds were evaluated by gas chromatography/mass spectrometry. Results: The DE showed a dose-dependent cytotoxicity in all the testes cell lines. The cell viability at doses of 250, 500, and 1000 μg/ml of DE was found as 87%, 75%, and 48% in HepG2; 95%, 85%, and 78% in A-549; and 77%, 51%, and 35% in MCF-7 cells, respectively. The GCMS analysis indicated the presence of 37 compounds. The fatty acids and esters, fatty alcohols, and steroid ester were predominant in the DE. The IC50 value of isolated pure compound (OA) was determined at 735.2 μg/ml in HepG2, 909.1 μg/ml in A549, and 675.6 μg/ml in MCF-7 cells. Conclusion: These results suggest that DE has promising anticancer potential and OA could be the compound contributing to cytotoxicity.