Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of activity against different pathogenic organisms and they represent promising new drugs to overcome the emergence of resistance to antibiotics in bacteria. (P)GKY20 is an antimicrobial peptide with a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. However, its mechanism of action is still unknown. Here, we use fluorescence spectroscopy and differential scanning calorimetry combined with atomic force microscopy to characterise the binding of (P)GKY20 with model biomembranes and its effect on the membrane's microstructure and thermotropic properties. We found that (P)GKY20 selectively perturbs the bacterial-like membrane via a carpet-like mechanism employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. These results shed a first light on the action mechanism of (P)GKY20 and could represent an important contribution to the development of new peptides serving as antimicrobial agents.