The current study intends to evaluate the cytotoxic activity of both 70% methanol extract and petroleum ether extract of Juniperus phoenicea L. aerial parts, as well as to analyze the chemical profile of both extracts. The result was affirmed that the hydroalcoholic extract possessed a very potent cytotoxicity against ovarian cancer cell line (SKOV-3) (IC50 =0.01 μg/ml) more than that of doxorubicin, a reference anticancer agent, (IC50 =0.95 μg/ml), and a potent cytotoxicity against prostate cancer cell line (PC-3) (IC50 =13.60 μg/mL). On the other hand, petroleum ether extract exhibited moderate cytotoxicity against SKOV-3 (IC50 = 29.6) and a weak effect against PC-3 cell lines (73.63 μg/ml). Moreover, the results revealed the identification of sixteen phenolic compounds in hydroalcoholic extract using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). Catechin, 3.4-dihydroxybenzoic acid, naringenin and gallic acid are presented as the major compounds. In addition, myricetin, quercetin, catechin, gallic acid and p-Coumaric acid were isolated and identified by different spectrometric methods. GC/MS analysis revealed the identification of seventeen compounds of the unsaponifiable matter of Juniperus phoenicea L. Where, nonacosanol, 1-(2-Octyldecyl) octahydropentalene and gamma-sitosterol are presented as the major compounds. On the other hand, fourteen compounds were identified from the saponifable matter. Hexadecanoic acid, 9-octadecenoic acid, and docosanoic acid were found to be the major constituents. These findings suggest that Juniperus phoenicea aerial parts may be an effective drug for the manipulation of different types of cancer after applying clinical trials. These outcomes open new areas for the alternative use of natural products in the drug discovery.