Aminoglycoside-modifying enzymes (AMEs) constitute the most prevalent mechanism of resistance to aminoglycosides by bacteria. We show that aminoglycosides can be doubly modified by the sequential actions of AMEs, with the activity of the second AME in most cases unaffected, decreased, or completely abolished. We demonstrate that the bifunctional enzyme AAC(3)-Ib/AAC(6')-Ib' can diacetylate gentamicin. Since single acetylation does not always inactivate the parent drugs completely, two modifications likely provide more-robust inactivation in vivo.