This study was conducted to evaluate the chemical composition, antioxidant and antibacterial activities of the essential oil of Pimpinella anisum L. seeds. The main constituents of the essential oil obtained by hydrodistillation using a Clevenger-type apparatus were identified by GC, GC/MS-EI, GC/MS-CI, and NMR spectroscopy. The antioxidant potential was assessed by using the DPPH method and the ferric reducing power. The antibacterial activity was determined by using the disk diffusion and the micro dilution methods against some Gram-positive and negative pathogenic bacteria. Results showed that aniseeds essential oil was characterized by a higher yield 2.6 ± 0.02% and good physic-chemical characteristics. Chemical analysis showed that the major components of the essential oil identified were anethole and estragole with percentages of 94.82 and 1.69%. Aniseeds essential oil showed a higher percentage of inhibition of DPPH 88.3 ± 0.5% and a lower value of IC50 118 ± 1.5 µg mL-1 determined at concentration of 1000 µg mL-1. This oil displayed a good ability to reduce Fe+3 to Fe+2 and provided an optical density of 1.78 ± 0.3 and IC50 of 60 ± 0.2 µg mL-1. Tested oil showed bactericidal activity against Pseudomonas aeruginosa and Escherichia coli with report MBC/MIC of 2 and antibacterial effect against Staphylococcus aureus with MBC/MIC of 32. It can be concluded that aniseeds essential oil contains substances with significant biological potential such as anethole and estragole that can be exploited in different pharmaceutical and therapeutic fields.