Our previous findings elucidated that the hydroethanolic extract of Echium angustifolium Mill. aerial parts and its defatted (polar) fraction supported by bio-guided fractionation possessed potential anticancer and antioxidant activities, as well as other documented plant therapeutic uses. Consequently, the present study aimed to evaluate the hexane (non-polar) extract of E. angustifolium aerial parts for in vitro anticancer, antioxidant, and anti-inflammatory activities accompanied by characterization of its bioactive constituents using GC-MS and LC-ESI-MS techniques in order to gain a deeper understanding of this medicinal plant that could be developed as a phytomedicine. This extract exhibited high inhibition against HCT116 (IC50 = 12 µg/mL) & HEPG2 (IC50 = 18 µg/mL) cancer cell lines, with antioxidant and anti-inflammatory potentials. GC-MS of the analyzed extract led to identifying 24 volatile constituents, among them, ethyl esters of palmitic acid and linoleic acid were the most abundant oxygenated compounds. Meanwhile, 10 nonvolatile components were annotated by LC-ESI-MS comprising five phenolic acid derivatives, two lignans, echimidine, acetylshikonin, and quinic acid. Moreover, some of these phytoconstituents were identified in this plant species for the first time. These results justify using non-polar E. angustifolium extract as a possible source of anticancer, antioxidant, and anti-inflammatory agents, which can be better confirmed by further in vivo studies.