Background: Acinetobacter is a serious nosocomial pathogen causing critical morbidity and mortality. The drug resistance of this organism is alarmingly high leaving few options for treatment. Numerous mechanisms are involved in its resistance to drug therapy. The active efflux mechanisms are an important factor for the development of multidrug-resistant Acinetobacter which is regulated by the AdeRS operon. The aim of this study end to determine the expression of AdeR and AdeS genes in multidrug‑resistant strains of Acinetobacter isolated from patients in Menoufia University Hospitals. Methods: This study included 100 strains of drug-resistant Acinetobacter spp., isolated from patients in Menoufia University Hospitals. They were collected from different clinical samples. Acinetobacter spp., strains were identified and their antibiotic susceptibilities were determined. Real-time PCR was performed to detect the expression of AdeR and AdeS genes. Results: The resistance of Acinetobacter spp., isolates to tested antibiotics were (94%) to piperacillin-tazobactam, (90%) to ampicillin-sulbactam, (80%) to ceftazidime, (55%) to levofloxacin, (50%) to amikacin, and (~52%) to carbapenems. The AdeR and AdeS genes were expressed in (84%) and (88%) of isolates, respectively. The AdeR and AdeS genes were expressed in (88.4%) and (90.7%) of multidrug-resistant strains, respectively. Conclusion: The majority of Acinetobacter spp., isolates are highly resistant to the most commonly used antibiotics. Also, high expression of adeRS genes may be responsible for the observed resistance among Acinetobacter isolates that demonstrate the possible role of efflux pump regulator genes in multidrug-resistant Acinetobacter.