Cardiolipin is an anionic tetra-acyl chained glycerophospholipid that increases lipid packing levels and induces intrinsic negative curvature in membranes. Cardiolipin is found in Staphylococcus aureus (S. aureus) membranes, where increased levels of this lipid are induced at the expense of diacyl phosphatidylglycerol in response to stress. We investigate cardiolipin as an inhibitor of the lytic activity of the cationic antimicrobial peptides LL-37 and ∆M2 in model systems with varying phosphatidylglycerol/cardiolipin ratios. Using HPTLC, we show that S. aureus (RN4220), under different growth conditions, has a phosphatidylglycerol/cardiolipin ratio of 80:20. From this, we chose three model systems to evaluate (100:0, 80:20, 60:40). ∆M2 presents higher binding affinity towards all mixtures compared to LL-37. This correlates with the higher antimicrobial activity of ∆M2 compared to LL-37 in S. aureus (MIC90 of 14 μM for ∆M2 and 57.7 μM for LL-37). Laurdan GP shows that Cardiolipin decreases lipid headgroup spacing. We find that cardiolipin does not affect ∆M2 or LL-37 binding to phosphatidylglycerol/cardiolipin liposomes. Instead, cardiolipin inhibits the ability of both peptides to induce calcein leakage in model liposomes. In conclusion, cardiolipin can reduce cAMP activity by inhibiting lysis but not binding.