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Background: Acinetobacter is one of the most important pathogens that causes high prevalence of infection in clinical centers. Natural products are major sources of new anti-infective agents and considered as an alternative medicine for the treatment of infectious diseases. Objective: This study aims to evaluate the antibacterial activity of aqueous and methanol extract of Artemisia annua on the clinically isolated Acinetobacter and the combination of the plant extract with amikacin and imipenem. Methods: The aqueous and methanol extract of the aerial parts of this plant was prepared by maceration. Antibacterial activity was determined by the Broth microdilution method. The presence of adeI and adeB genes in Acinetobacter isolates was investigated. The combination effects of antibiotics were performed using the checkerboard method. The effect of plant extract on bacterial efflux pumps was also investigated. Results: MIC values of the methanol extract of A. annua were from 4.46 mg/ml to 8.92 mg/ml. The effects of the interaction of the plant extract with the amikacin and imipenem represent a significant synergistic influence. Findings indicated that the plant extract was able to inhibit the efflux pump remarkably. Conclusion: The combination of the plant extract with amikacin and imipenem can be used in the treatment of infections caused by antibiotic-resistant Acinetobacter and probably other infectious diseases.