Background The emergence of carbapenemases in Enterobacteriaceae is driving a search for therapeutic alternatives. We tested ACHN-490, a sisomicin derivative that evades all plasmid-mediated aminoglycoside-modifying enzymes, against 82 carbapenem-resistant Enterobacteriaceae isolates. Comparators included internationally and locally available aminoglycosides. Methods The isolates variously had KPC (n?=?12), SME-1 (n?=?1), IMP (n?=?13), VIM (n?=?5), NDM (n?=?17) or OXA-48 (n?=?19) carbapenemases, or had combinations of impermeability with AmpC (n?=?5) or extended-spectrum ß-lactamases (n?=?10). They included 53 Klebsiella spp., 19 Enterobacter spp., 6 Escherichia coli and 4 others; most were multiresistant. Genes were identified by PCR and sequencing; MICs were measured by CLSI agar dilution. Results ACHN-490 was active at =2 mg/L against all 65 isolates with carbapenem resistance mechanisms other than NDM enzyme, mostly with MICs of 0.12–0.5 mg/L; isepamicin was active against 63/65 at =8 mg/L. In contrast, 35% were resistant to gentamicin at 4 mg/L, 61% to tobramycin at 4 mg/L and 20% to amikacin at 16 mg/L. However, 16 of the 17 isolates with NDM-1 enzyme were resistant to ACHN-490, with MICs =64 mg/L, and these were cross-resistant to all other human-use aminoglycosides tested. Their behaviour was associated with ArmA and RmtC 16S rRNA methylases. Apramycin (a veterinary aminoglycoside) retained its full activity, with MICs of 4–8 mg/L versus strains with armA or rmtC, though resistance was seen in one Klebsiella pneumoniae with AAC(3)-IV (MIC =256 mg/L). Conclusions ACHN-490 has potent activity versus carbapenem-resistant isolates, except those also harbouring 16S rRNA methylases; isepamicin is also widely active, though less potent than ACHN-490. Evasion of 16S rRNA methylases by apramycin is noteworthy and may provide a starting point for future aminoglycoside development.