The present study aimed to investigate Senecio anteuphorbium, an endemic plant growing in West Morocco and widely used in local folk medicine. The essential oil (EO) extracted from the aerial parts was analyzed by gas chromatography and tested for allelopathic activity. The quantitation of the volatiles was carried out by means of GC-FID with response factors, which were validated through reliable calibration procedures, based on external and internal standardization. This analytical approach allowed to define the real concentration of each constituent (weight%, g/100 g) alongside the conventional relative percent. On the other hand, the identification process was supported by a dual matching based on both mass spectra and retention indices. The essential oil resulted in being rich in sesquiterpenes, with the predominant constituents being bicyclogermacrene (22.75 g/100 g), spathulenol (25.26 g/100 g), epi-γ-eudesmol (6.8 g/100 g), and selina-4,11-diene (5.08 g/100 g). The allelopathic effect was evaluated by studying the inhibition of the germination and growth of Lactuca sativa seeds. A potent allelopathic effect was recorded by the essential oil at a dose of 0.281 mg/mL, with almost a total inhibition of germination.