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Ocimum basilicum is a valuable source of bioactive metabolites with high preventive and therapeutic effectiveness. Here we aimed to investigate the effect of phenological stages (vegetative and flowering stages) on essential oil composition and biological activities of two varieties of O. basilicum (Fino Verde variety and Genovese varieties). To this end, the level of essential oils, flavonoids and phenols, as well as antioxidant and antifungal activities were measured. At the metabolic level, essential oil at vegetative stage of O. bailicum Fino Verde and Genovese variety was constituted by 22 and 26 compounds representing 71.68% and 82.54% of the total oil, respectively. Where germacrene D (10.07%), bicyclogermacrene (6.07%) and β-elemene (4.88%) were the most present components in Fino Verde variety. Moreover, 22.19% are oxygenated monoterpenes represented mainly by the linalool (15.18%) and 1.8 cineole (6.36%) in Genovese variety. The individuals of essential oils were significantly increased to 40 components in Fino Verde variety (98.01% of total essential oil) and decreased to 15 components (95.6% of total essential oil) in Genovese variety at flowering stage. At this stage, the oxygenated monoterpenes (78.4%) were the major fraction represented by linalool (40.1%) and 1.8 cineole (30.96%) in Fino Verde variety, however 64.69% were esters which mainly represented by the methyl cinnamate (64.69%), and 16.83% of oxygenated monoterpenes and Linalool (12.7%) were recorded for Genovese variety. Genovese variety showed the highest levels at both vegetative and flowering stage compared to Fino Verde variety. At flowering stage, the two varieties showed high antioxidant and antifungal activities. Overall, O. basilicum properties offer prospects for their use as a source, particularly at flowering stage to extend new medicines based on natural bioactive molecules.