16S rRNA methylases confer high-level resistance to aminoglycosides which are used to treat serious infections caused by gram-negative bacteria, such as Acinetobacter spp. Some genes encoding these enzymes are disseminated worldwide, while others were detected in only some countries. The objective was to characterize the susceptibility profile to aminoglycosides (amikacin and gentamicin) of clinical isolates of Acinetobacter spp. from an oncological hospital in Recife, and given the resistance to both antimicrobials, to characterize minimal inhibitory concentrations (MICs) of amikacin, gentamicin and tobramycin, the occurrence of 16S rRNA methylase genes (armA, rmtB, rmtC and rmtD) and of ß-lactamase gene (blaKPC) and the clonal profile. Isolates resistant to both antimicrobials, amikacin and gentamicin, were selected by disk diffusion technique in Mueller-Hinton agar and identified. Broth microdilution was conducted to determine MICs of amikacin, gentamicin, and tobramycin. These isolates were subjected to polymerase chain reaction and pulsed-field gel electrophoresis. Among 23 analyzed isolates, 12 (52.2%) were resistant to gentamicin and amikacin and identified as Acinetobacter baumannii. Among these, 11 (91.7%), 12 (100%), and 9 (75%) isolates showed respectively MICs > 256 µg/mL of amikacin, > 64 µg/mL of gentamicin, and > 64 µg/mL of tobramycin. The armA gene was found in 12 (100%) isolates and 6 (50%) showed coexistence of armA, rmtB, and rmtC genes. The rmtD and blaKPC genes were not detected. These isolates showed high genetic similarity (92%) and were classified as clone A. Elaboration and fulfillment of measures are thus essential to prevent the spread of this resistance mechanism.