This study deals with the structural perturbations that the aminoglycoside antibiotic gentamicin (GENT) can produce to phospholipid bilayers. Two multi-bilayer systems, one built-up of dimyristoylphosphatidylcholine (DMPC) and the other of dimyristoylphosphatidylethanolamine (DMPE) were allowed to interact with GENT. The experiments were performed in both a hydrophobic and a hydrophilic medium below the phospholipid main transition temperatures. X-ray diffraction techniques were used to determine the extent of the perturbation induced by GENT. The maximum effect was attained when GENT interacted with DMPC in the hydrophobic medium. On the other hand, GENT in aqueous solutions was unable to perturb in any significant extent the structure of the phospholipids under study.