The widespread emergence of bacterial resistance has led to an urgent need to develop new strategies to regain the efficacy of antibacterials. One of the emerging concept is to target the bacterial membrane bilayer. Aminoglycosides are among the most potent antimicrobials to treat severe infections. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine in the aim to obtain amphiphilic antibiotics able to disturb bacterial membrane bilayer. One to four hydroxyl functions of neamine were capped with phenyl, naphthyl, pyridyl, or quinolyl rings. The 3',4'-, 3',6- and the 3',4',6-2-naphthylmethylene (2NM) derivatives were active against both sensitive and resistant S. aureus strains. The trisubstituted derivative, also showed marked antibacterial activity against Gram (-) bacteria, including resistant strains (1). Regarding its mechanism of action, it showed only a weak and aspecific binding to a model bacterial 16S rRNA as well as a lower ability to decrease 3H leucine incorporation into proteins in P.aeruginosa, suggesting it acts through a mechanism probably involving membrane destabilization. To understand the molecular mechanism involved, we determined the ability of 3’,4’,6-tri-2NM neamine to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using Atomic Force Microscopy (AFM), we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3’,4’,6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3’,6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that 3’,4’,6-tri-2NM neamine derivatives target the membrane of P. aeruginosa (2). This should offer promising prospects in the search for new antibacterials against resistant drug or biocide strains.