Antimicrobial peptides (AMPs) raise a great interest as an antibiotic alternative, yet their unpredictable side effects that originate from their low specificity hinder a major success as a therapeutic agent. How can we lower the peptide dosage to avoid the cytotoxicity without sacrificing their antimicrobial effects? Scientists have reported that bacteria were killed much more efficiently when two human AMPs, defensin and cathelicidin, are mixed. In this work, we report that the same AMP mixture unexpectedly suppressed the cytotoxicity when incubated with eukaryotic cells. These discoveries suggest that we can “double-benefit” from mixing peptides for broadening the therapeutic window by orders of magnitudes. However, how an identical couple of peptides kills bacteria more efficiently yet reduces the cytotoxicity is left as a mystery. To tackle this open question, we characterized the cooperative function between defensin and cathelicidin, in living cells (MDCK, HUVEC) and in synthetic bilayer systems by calcium sensitive dyes, mitochondrial staining, single channel conductance measurements, electrochemical impedance spectroscopy (EIS), quartz crystal microbalance with dissipation (QCM-D), fluorescence recovery after photobleaching (FRAP), isothermal titration calorimetry (ITC) and circular dichroism (CD). To our best knowledge, this is the first report on the inhibitory interference between defensin and cathelicidin and their mechanistic investigation at the molecular level.