Jelleines are four naturally occurring peptides that comprise approximately eight or nine C-terminal residues in the sequence of the major royal jelly protein 1 precursor (Apis mellifera). The difference between these peptides is limited to one residue in the sequence, but this residue has a significant impact in their efficacy as antimicrobials. In peptide-bilayer experiments, we demonstrated that the lytic, pore-forming activity of Jelleine-I is similar to that of other cationic antimicrobial peptides, which exhibit stronger activity on anionic bilayers. Results from molecular dynamics simulations suggested that the presence of a proline residue at the first position is the underlying reason for the higher efficacy of Jelleine-I compared with the other jelleines. Additionally, simulations suggested that Jelleine-I tends to form aggregates in water and in the presence of mimetic membrane environments. Combined experimental evidence and simulations showed that the protonation of the histidine residue potentiates the interaction with anionic palmitoyl-oleoyl-phosphatidylcholine/palmitoyl-oleoyl-phosphatidylglycerol (POPC/POPG) (70:30) bilayers and reduces the free energy barrier for water passage. The interaction is driven by electrostatic interactions with the headgroup region of the bilayer with some disturbance of the acyl chain region. Our findings point to a mechanism of action by which aggregated Jelleine-I accumulates on the headgroup region of the membrane. Remaining in this form, Jelleine-I could exert pressure to accommodate its polar and nonpolar residues on the amphiphilic environment of the membrane. This pressure could open pores or defects, could disturb the bilayer continuity, and leakage would be observed. The agreement between experimental data and simulations in mimetic membranes suggests that this approach may be a valuable tool to the understanding of the molecular mechanisms of action.