Amongst the many synthetic aminoglycoside analogues that were developed to regain the efficacy of this class of antibiotics against resistant bacterial strains, the 1-N-acylated analogues are the most clinically used. In this study we demonstrate that 6'-N-acylation of the clinically used compound tobramycin and 6'''-N-acylation of paromomycin result in derivatives resistant to deactivation by 6'-aminoglycoside acetyltransferase (AAC(6')) which is widely found in aminoglycoside resistant bacteria. When tested against AAC(6')- or AAC(3)-expressing bacteria as well as pathogenic bacterial strains, some of the analogues demonstrated improved antibacterial activity compared to their parent antibiotics. Improvement of the biological performance of the N-acylated analogues was found to be highly dependent on the specific aminoglycoside and acyl group. Our study indicates that as for 1-N-acylation, 6'- and 6'''-N-acylation of aminoglycosides offer an additional promising direction in the search for aminoglycosides capable of overcoming infections by resistant bacteria.