Amphiphilic aminoglycoside antimicrobials are an emerging class of new antibacterial agents with novel modes of action. Previous studies have shown that amphiphilic neomycin-B and kanamycin-A analogs restore potent antibacterial activity against Gram-positive neomycin-B- and kanamycin-A-resistant organisms. In this paper, we investigated the antibacterial properties of a series of amphiphilic tobramycin analogs. We prepared tobramycin-lipid conjugates, as well as tobramycin-peptide triazole conjugates, and studied their antibacterial activities against a panel of Gram-positive and Gram-negative bacterial strains, including isolates obtained from Canadian hospitals. Our results demonstrate that the antibacterial activity of amphiphilic tobramycin is greatly affected by the length and nature of the hydrophobic lipid tail, whereas the nature of the polycationic headgroup or the number of cationic charges appear to be less important. Replacement of the hydrophobic tail by a fluorinated lipid confers good activity against two Pseudomonas strains and reduces hemolytic activity. However, susceptibility studies in the presence of bovine serum albumin indicate that all amphiphilic tobramycin analogs are strongly protein-bound, leading to a typical four- to eight-fold increase in MIC.