The aim of this work was to study the chemical composition, antioxidant properties and antibacterial activity of Tunisian fennel (Foeniculum vulgare Mill.) leaves grown under conventional and organic cultivation conditions. Analysis of chemical composition of fennel leaves showed high sugar contents whatever the cultivation conditions and the tested cultivar, and ranged from 2.7 to 7.39%. Protein contents varied from 5.94 to 7.82 and from 4.41 to 7.15% under conventional and organic cultivation conditions, respectively. Moreover, the chemical composition of the fennel leaves essential oils (EOs) showed high levels of limonene and trans-anethole. Data analysis of EOs antioxidant activity showed that E1 and E4 cultivars grown under conventional and organic cultivation conditions, respectively, had the lowest IC50 with mean values of 0.052 and 0.045 mg/mL, respectively. Furthermore, antibacterial activity of fennel leaves EOs was shown to be effective against Listeria monocytogenes with an inhibition diameter of 30 mm with a concentration of 100 mg/mL for the EOs extracted from the E3 cultivar cultivated under conventional conditions.