Abstract: Neroli essential oil is extracted from the fragrant blossoms of the bitter orange tree. It is one of the most widely used floral oils in perfumery. In this study chemical composition and in vitro antimicrobial and antioxidant activities of neroli oil are investigated. The essential oil of fresh Citrus aurantium L. Flowers (Neroli oil) cultivated in North East of Tunisia (Nabeul) were analyzed by GC-FID and GC-MS. About 33 compounds were identified, representing 99% of the total oil. Limonene (27.5%) was the main component followed by (E)-nerolidol (17.5%), α-terpineol (14%), α-terpinyl acetate (11.7%) and (E, E)-farnesol (8%). Antimicrobial activity was determined by Agar-well-diffusion method against 6 bacteria (3 Gram-positive and 3 Gram-negative), 2 yeasts and 3 fungi. Neroli oil exhibited a marked antibacterial activity especially against Pseudomonas aeruginosa. Moreover, Neroli oil exhibited a very strong antifungal activity compared with the standard antibiotic (Nystatin) as evidenced by their inhibition zones. Antioxidant activity determined by ABTS assay showed IC50 values of 672 mg L-1. Finally, this study may be considered as the first report on the biological properties of this essential oil. The results of this study have provided a starting point for the investigations to exploit new natural substances present in the essential oil of C. aurantium L. flowers.