To evaluate the in vivo effectiveness of a combination treatment containing ranalexin (a natural antimicrobial peptide) and lysostaphin (an antistaphylococcal endopeptidase) for reducing nasal burden of methicillin-resistant Staphylococcus aureus (MRSA). The community-acquired MRSA strain S. aureus NRS384 (USA300-0114) was used in the present study because it is commonly isolated from human nares and it established consistent and reproducible colonization of cotton rat nares. This model was used to evaluate the efficacy of ranalexin/lysostaphin gels (0.1%-1% w/v; administered intranasally once or once per day for 3 consecutive days) for reducing nasal MRSA burden. Control animals were administered vehicle gel only (0.5% hydroxypropyl methylcellulose) or 2% mupirocin, which is used clinically for nasal decolonization of MRSA. Nasal MRSA burden was assessed at 192 h post-inoculation, which was at least 72 h after the final treatment had been administered. An additional study assessed the efficacy of 0.1% ranalexin/lysostaphin against a mupirocin-resistant MRSA strain (MUP20), which had been selected by serial passage of S. aureus NRS384 through subinhibitory concentrations of mupirocin. Gels containing 0.1% ranalexin/lysostaphin consistently reduced median nasal burden of MRSA to an extent similar to or greater than 2% mupirocin. Treatment with 0.1% ranalexin/lysostaphin was also effective against the MUP20 strain. There was evidence for only minimal irritancy in cotton rat nares administered three doses of 0.1% ranalexin/lysostaphin, suggesting that this agent is suitable for short-course therapy such as is employed currently for nasal decolonization with mupirocin. Ranalexin/lysostaphin could serve as an alternative to mupirocin for nasal decolonization of MRSA.