A short site-selective strategy for the activation and derivatization of alcohols of the clinically important aminoglycoside tobramycin is reported. The choice of amine protecting group affected the site-selective conversion of secondary alcohols of tobramycin into leaving groups. Temperature-dependent, chemoselective sequential nucleophilic displacements resulted in hetero- and homodithioether tobramycin-based cationic amphiphiles that demonstrated marked antimicrobial activity and impressive membrane selectivity.