Nasal colonization of Staphylococcus aureus is a risk factor for pathogenic autoinfection, particularly in postoperative patients and the immunocompromised. As such, standardized preoperative nasal decolonization of S. aureus has become a major consideration for the prevention of nosocomial infection. However, only a few treatment options for nasal decolonization are currently available, with resistance to these approaches already a concern. Here we have identified the macrocyclic θ-defensin analogue RC-101 as a promising anti-S. aureus agent for nasal decolonization. RC-101 exhibits bactericidal effects against S. aureus with the use of in vitro epithelium-free systems, while also preventing the pathogen's proliferation and attachment in an ex vivo human nasal epithelial cell adhesion model and an organotypic model of human airway epithelia. Peptide concentrations as low as 2.5 μM elicited significant reductions in S. aureus growth in epithelium-free systems, with 10 μM concentrations being completely bactericidal for all strains tested, including USA300. In ex vivo nasal colonization models, RC-101 significantly reduced adherence, survival, and proliferation of S. aureus on human nasal epithelia. Reductions in S. aureus viability were evident in these assays, with as little as 1 μg of peptide per tissue, while 10 μg of RC-101 completely prevented adhesion of all strains tested. Furthermore, RC-101 did not exhibit cellular toxicity to human nasal epithelia at concentrations up to 200 μM, nor did it induce a proinflammatory response in these cells. Collectively, the findings of this study identify RC-101 as a potential preventative of S. aureus nasal colonization.