The antimicrobial peptide GL13K encompasses 13 amino acid residues and has been designed and optimized from the salivary protein BPIFA2 to exhibit potent bacteriocidal and anti-biofilm activity against Gram-negative and Gram-positive bacteria as well as anti-lipopolysaccharide activity in vitro and in vivo. Here, the peptide was analyzed in a variety of membrane environments by circular dichroism spectroscopy and by high-resolution multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. Whereas in the absence of membranes a random coil conformation predominates, the peptide adopts a helical structure from residue 5 to 11 in the presence of dodecylphosphocholine micelles. In contrast, a predominantly β-sheet structure was observed in the presence of lipid bilayers carrying negatively charged phospholipids. Whereas 15N solid-state NMR spectra are indicative of a partial alignment of the peptide 15N-1H vector along the membrane surface, 2H and 31P solid-state NMR spectra indicate that in this configuration the peptide exhibits pronounced disordering activities on the phospholipid membrane, which is possibly related to antimicrobial action. GL13K, thus, undergoes a number of conformational transitions, including a random coil state in solution, a helical structure upon dilution at the surface of zwitterionic membranes, and β-sheet conformations at high peptide:lipid ratios.