Novel approaches targeting the host's immune response to treat Staphylococcus aureus infections have significant potential to improve clinical outcomes, in particular during infection with antibiotic-resistant strains. The hyaluronic acid-binding peptide (HABP) PEP35 was assessed for its ability to treat S. aureus infections using a clinically relevant murine model of surgical wound infection. PEP35 demonstrated no direct antimicrobial activity against a range of antibiotic-susceptible and antibiotic-resistant clinical isolates of Staphylococcus aureus. However, when this peptide was administered at the onset of infection and up to 4 h postchallenge with a methicillin-susceptible (MSSA) or a methicillin-resistant (MRSA) strain of S. aureus, it significantly reduced the bacterial burden at the wound infection site. PEP35 reduced the tissue bacterial burden by exclusively modulating the local neutrophil response. PEP35 administration resulted in a significant early increase in local CXCL1 and CXCL2 production, which resulted in a more rapid influx of neutrophils to the infection site. Importantly, neutrophil influx was not sustained after treatment with PEP35, and administration of PEP35 alone did not induce a local inflammatory response. The immunomodulatory effects of PEP35 on CXC chemokine production were TLR2 and NF-κB dependent. We propose a novel role for a HABP as an innate immunomodulator in the treatment of MSSA and MRSA surgical wound infection through enhancement of the local CXC chemokine-driven neutrophil response.