INTRODUCTION: This study aimed to determine the role of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase (ArmA) in Acinetobacter baumannii clinical isolates. METHODS: We collected 100 clinical isolates of A. baumannii and identified and confirmed them using microbiological tests and assessment of the OXA-51 gene. Antibiotic susceptibility testing was carried out using disk agar diffusion and micro-broth dilution methods. The presence of AME genes and ArmA was detected by PCR and multiplex PCR. RESULTS: The most and least effective antibiotics in this study were netilmicin and ciprofloxacin with 68% and 100% resistance rates, respectively. According to the minimum inhibitory concentration test, 94% of the isolates were resistant to gentamicin, tobramycin, and streptomycin, while the highest susceptibility (20%) was observed against netilmicin. The proportion of strains harboring the aminoglycoside resistance genes was as follows: APH (3′)- VIa ( aph A6) (77%), ANT (2”)- Ia ( aad B) (73%), ANT (3”)- Ia ( aad A1) (33%), AAC (6′)- Ib ( aac A4) (33%), ArmA (22%), and AAC (3)- IIa ( aac C2) (19%). Among the 22 gene profiles detected in this study, the most prevalent profiles included APH (3′)- VIa + ANT (2”)- Ia (39 isolates, 100% of which were kanamycin-resistant), and AAC (3)- IIa + AAC (6′)- Ib + ANT (3”)- Ia + APH (3′)- VIa + ANT (2”)- Ia (14 isolates, all of which were resistant to gentamicin, kanamycin, and streptomycin). CONCLUSIONS: High minimum inhibitory concentration of aminoglycosides in isolates with the simultaneous presence of AME- and ArmA-encoding genes indicated the importance of these genes in resistance to aminoglycosides. However, control of their spread could be effective in the treatment of infections caused by A. baumannii.