An antibacterial peptide, Magainin I, was covalently bound to a mixed 11-mercaptoundecanoïc acid (MUA) and 6-mercaptohexanol (C6OH) (ratio 1:3) Self-Assembled Monolayer (SAM) on gold surfaces. Each step of the surface functionalization was characterized by Polarization Modulation Reflection Absorption InfraRed Spectroscopy (PM-RAIRS) and X-ray Photoelectron Spectroscopy (XPS). The antibacterial activity of the anchored Magainin was tested against three Gram-positive bacteria (Listeria ivanovii, Enterococcus faecalis and Staphylococcus aureus), and the results revealed that the adsorbed Magainin I reduced by more than 50% the adhesion of bacteria at the surface, together with the killing of the bacteria that nonetheless adhered to the surface. No release of the peptide was observed upon contact with the bacterial suspension; the activity has proven to be persistent overtime, up to six months after the first use.