Invasive species are considered as one of the major threats to ecosystems worldwide. Although invasive plants are regarded as a foe, they could be considered as natural resources for valuable bioactive compounds. The present study aimed to characterize the chemical composition of the essential oil (EO) from the invasive plant Argemone ochroleuca Sweet, collected from Saudi Arabia, as well as to evaluate its phytotoxic activity. Seventy-four compounds were characterized via GC-MS analysis of EO representing 98.75% of the overall mass. The oxygenated constituents (79.01%) were found as the main constituents, including mono- (43.27%), sesqui- (17.67%), and di-terpenes (0.53%), as well as hydrocarbons (16.81%) and carotenoids (0.73%). Additionally, 19.69% from the overall mass was characterized as non-oxygenated compounds with mono- (1.77%), sesquiterpenes (17.41%), and hydrocarbons (0.56%) as minors. From all identified constituents, trans-chrysanthenyl acetate (25.71%), γ-cadinene (11.70%), oleic acid, methyl ester (7.37%), terpinene-4-ol (4.77%), dihydromyrcenol (2.90%), α-muurolene (1.77%), and γ-himachalene (1.56%) were found as abundant. The EO of A. ochroleuca showed significant phytotoxic activity against the test plant Lactuca sativa and the noxious weed Peganum harmala. The EO attained IC50 values of 92.1, 128.6, and 131.6 µL L−1 for seedling root growth, germination, and shoot growth of L. sativa, respectively, while it had IC50 values of 134.8, 145.7, and 147.9 µL L−1, respectively, for P. harmala. Therefore, this EO could be used as a bioherbicide against weeds, while further study is recommended for the characterization of the authentic materials of the main compounds in the EO as well as for the evaluation of potency of this oil on a field scale and the determination of its biosafety.