Thousands of antimicrobial peptides have been observed and studied in the past decades; however, their membrane-active mechanisms are ambiguous due to their dynamic structure in the cell membrane. Here, we applied both molecular dynamics (MD) simulations and biophysical experiments to study the small membrane-active antimicrobial peptide Hylaseptin P1 (HSP1), which has significant selectivity towards anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and bacterial model membranes. HSP1 does not bind and fold onto human red blood cell model membranes, and it only binds, but does not fold, in zwitterionic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) membranes. This suggests that the lipid chemistry and membrane rigidity are key to prevent HSP1 binding onto membranes, and the lipid headgroup charge may further promote peptide folding in the membrane. Our experiment-validated MD simulations suggest a carpet-like model mechanism for HSP1 through peptide binding, folding, aggregation, and assembly. HSP1 is shorter than the membrane thickness; therefore, the folded peptides aggregate on the surface, cross the membrane, and the oligomeric structure is supported by several surface-bound peptides in both bilayer leaflets.