<jats:p>Insects respond to a bacterial challenge by rapidly synthesizing a diverse range of antibacterial and antifungal peptides. One of them, drosocin, a 19‐residue prone‐rich antibacterial peptide, was isolated from <jats:italic>Drosophila.</jats:italic> This peptide carries a disaccharide moiety attached to a threonine residue in mid‐chain position. The present report describes the enlarged‐scale chemical synthesis of drosocin, glycosylated with Gal(β1→3)GalNAc(αL→O). We have studied the range of activity of the synthetic glycopeptide, of two truncated glycosylated isoforms, and of the unglycosylated L and D enantiomers. Both isolated and chemically synthesized drosocins carrying the disaccharide display the same antibacterial activity. Using circular dichroic spectroscopy we demonstrated that the O‐nked disaccharidic motif did not affect the backbone conformation of drosocin. The antibacterial activity of the synthetic glycopeptide was directed against gram‐negative strains with the exception of the gram‐positive bacteria <jats:italic>Micrococcus luteus.</jats:italic> Deletion of the first five N‐terminal residues completely aboshed the activity of drosocin. As a first approach to the study of the mode of action of drosocin, we have synthesized a non‐glycosylated d enantiomer and, using this molecule, we have shown that drosocin may act on the gram‐negative bacteria through a stereospecific target.