Asphodelus are among many plant species used in traditional medicine, they are used by native peoples for various pathologies. Asphodelus aestivus Brot. is one of Asphodelus species which is considered an important wild medicinal plant growing mainly at Mediterranean region. The present study aims to investigate the chemical constituents and cytotoxic activity of A. aestivus flower. Petroleum ether extract was analyzed by GC/MS assay which revealed the characterization of twenty two compounds, representing 97.14% of total extract. Also, the chromatographic investigation of the aqueous methanol extract led to the isolation of ten phenolics, from which, five compounds have been isolated for the first time from A. aestivus. They were identified as kaempferol (1), kaempferol 3-O-glucoside (2), kaempferol 3,7-di-O-glucoside (3), luteolin 7-O-glucoside )4), isoorientin (5), isoorientin 7-O-glucoside (lutonarin) (6), caffeic acid (7) emodin (8), apigenin 7-O-glucoside (9) and isovitexin-7-O-glucoside (saponarin) (10). Furthermore, LC-ESI-MS chemical profiling managed to detect the minor contents and led to the annotation of thirteen additional metabolites. In addition, the petroleum ether and aqueous methanol extracts were investigated against four human carcinoma cell lines; HepG2, HCT116, MCF7 and A549 at concentration of 100 mg/mL. The results showed moderate % of inhibition against MCF7 (63.8 and 68.4) followed by HEPG2 (52.3 and 65.7), respectively. Both extracts showed weak inhibition (5.7-14.3%) on HCT-116 and A549. To the best of our knowledge, this is considered the first report of the chemical analysis and biological evaluation of A. aestivus flowers.