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Antimicrobial resistance is increasing around the world and the search for effective treatment options, such as new antibiotics and combination therapy is urgently needed. The present study evaluates oregano essential oil (OEO) antibacterial activities against reference and multidrug-resistant clinical isolates of Acinetobacter baumannii (Ab-MDR). Additionally, the combination of the OEO and polymyxin B was evaluated against Ab-MDR. Ten clinical isolates were characterized at the species level through multiplex polymerase chain reaction (PCR) for the gyrB and blaOXA-51-like genes. The isolates were resistant to at least four different classes of antimicrobial agents, namely, aminoglycosides, cephems, carbapenems, and fluoroquinolones. All isolates were metallo-β-lactamase (MβL) and carbapenemase producers. The major component of OEO was found to be carvacrol (71.0%) followed by β-caryophyllene (4.0%), γ-terpinene (4.5%), p-cymene (3,5%), and thymol (3.0%). OEO showed antibacterial effect against all Ab-MDR tested, with minimum inhibitory concentrations (MIC) ranging from 1.75 to 3.50 mg mL−1. Flow cytometry demonstrated that the OEO causes destabilization and rupture of the bacterial cell membrane resulting in apoptosis of A. baumannii cells (p < 0.05). Synergic interaction between OEO and polymyxin B (FICI: 0.18 to 0.37) was observed, using a checkerboard assay. When combined, OEO presented until 16-fold reduction of the polymyxin B MIC. The results presented here indicate that the OEO used alone or in combination with polymyxin B in the treatment of Ab-MDR infections is promising. To the best of our knowledge, this is the first report of OEO and polymyxin B association against Ab-MDR clinical isolates. [ABSTRACT FROM AUTHOR]