Membrane-active peptides are useful tools in the design of multifunctional molecules. For example, peptide chimeras may release, after proteolysis of membrane-adsorbed molecules, pharmacologically active fragments. In previous work, Chim2, an antimicrobial peptide composed of a membrane-active module, an enzymatic hydrolysis site, and an agonist moiety for type 2 formyl peptide receptors (FPR2), was conceptualized. Based on Chim2, a peptide named Chim3 was designed, adding a consensus sequence for the bacterial signal peptidase I (Spase I). Spase I is a protease located in an extracytoplasmic face of Gram-positive and Gram-negative bacterial membranes and is essential for protein export. Chim3 was synthesized and its activity as an antimicrobial agent was determined. In addition, Chim3 was incubated with Escherichia coli and Staphylococcus aureus, and peptide hydrolysis products were evaluated by LC-MS/MS. Data demonstrate that Chim3 has potent antimicrobial activity. After incubation with bacteria, Chim3 underwent intense hydrolysis. Proteolysis was detected in the Chim3 Spase I consensus sequence after incubation with both bacteria, and the release of the FPR2 agonist segment was observed. The synthesis of an improved structure of Chim3 with N-methyl tyrosine in the FPR2 agonist segment was performed, resulting in CHIM3Y-NMe. This modification caused significantly higher concentrations of the FPR2 agonist portion arising from the modified peptide after incubation assays with E. coli. The modified FPR2 agonist WK(Y-NMe)M-NH