Citrus aurantium L. essential oil is commonly used as a flavouring agent. In the present study, the essential oil of fresh Citrus aurantium L. (CaEO) flowers cultivated in North East of Tunisia (Nabeul) was analyzed by GC-FID and GC-MS. 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%). The antimicrobial activity of the CaEO was evaluated against a panel of 13 bacteria and 8 fungal strains using agar diffusion and broth microdilution methods. Results have shown that the CaEO exhibited moderate to strong antimicrobial activity against the tested species. The investigation of the mode of action of the CaEO by the time-kill curve showed a drastic bactericidal effect after 5 min using a concentration of 624 μg/ml. The antioxidant activities of the CaEO were assayed by DPPH and beta carotene tests. Results showed that CaEO displayed an excellent DPPH scavenging ability with an IC₅₀ of 1.8 μg/ml and a strong Beta-carotene bleaching inhibition after 120 min of incubation with an IC₅₀ of 15.3 μg/ml. The results suggested that the CaEO possesses antimicrobial and antioxidant properties, and is therefore a potential source of active ingredients for food and pharmaceutical industry.