Aminoglycoside antibiotics are pseudosaccharides decorated with ammonium groups that are critical for their potent broad-spectrum antibacterial activity. Despite over three decades of speculation whether or not modulation of pKa is a viable strategy to curtail aminoglycoside kidney toxicity, there is a lack of methods to systematically probe amine-RNA interactions and resultant cytotoxicity trends. This study reports the first series of potent aminoglycoside antibiotics harboring fluorinated N1-hydroxyaminobutyryl acyl (HABA) appendages for which fluorine-RNA contacts are revealed through an X-ray cocrystal structure within the RNA A-site. Cytotoxicity in kidney-derived cells was significantly reduced for the derivative featuring our novel β,β-difluoro-HABA group, which masks one net charge by lowering the pKa without compromising antibacterial potency. This novel side-chain assists in evasion of aminoglycoside-modifying enzymes, and it can be easily transferred to impart these properties onto any number of novel analogs.