The in vitro activity of plazomicin was evaluated against 300 multidrug resistant (MDR) (carbapenemase and/or ESBL-producing) isolates from four hospitals in Athens, an area where carbapenemase-producing organisms are endemic. Most of the isolates were also resistant to the legacy aminoglycosides with the MIC(50)/MIC(90) to tobramycin, amikacin and gentamicin being 32/>32, 32/>32 and 4/>8 μg/ml, respectively. ACHN-490 retained activity (MICs4 μg/ml) against all isolates of Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp. tested with MIC(50) and MIC(90) of 1 and 2 μg/ml, respectively, irrespective of their MDR phenotype and it represents a promising alternative for the treatment of the most problematic Gram-negative pathogens.