Volatile oils (VOs) composition of plants is affected by several exogenous and endogenous factors. Male and female plants of the dioecious species exhibit variation in the bioactive constituents’ allocation. The chemical variation in the VOs between male and female plants is not well studied. In the present study, the chemical characterization of the VOs extracted from aerial parts of male and female ecospecies of Ochradenus arabicus was documented. Additionally, the extracted VOs were tested for their allelopathic activity against the weed Dactyloctenium aegyptium. Via GC-MS analysis, a total of 53 compounds were identified in both male and female plants. Among them, 49 compounds were identified from male plants, and 47 compounds were characterized in female plants. Isothiocyanates (47.50% in male and 84.32% in female) and terpenes (48.05% in male and 13.22% in female) were the main components of VOs, in addition to traces of carotenoid-derived compounds and hydrocarbons. The major identified compounds of male and female plants are m-tolyl isothiocyanate, benzyl isothiocyanate, butyl isothiocyanate, isobutyl isothiocyanate, carvone, and α-bisabolol, where they showed variation in the concentration between male and female plants. The O. arabicus VOs of the male plants attained IC50 values of 51.1, 58.1, and 41.9 μL L−1 for the seed germination, seedling shoot growth, and seedling root growth of the weed (D. aegyptium), respectively, while the females showed IC50 values of 56.7, 63.9, and 40.7 μL L−1, respectively. The present data revealed that VOs composition and bioactivity varied significantly with respect to the plant gender, either qualitatively or quantitatively.