Chronic rhinosinusitis (CRS) is a persistent inflammation of the sinus mucosa. Recalcitrant CRS patients are unresponsive to medical and surgical interventions and often present with nasal polyps, tissue eosinophilia, and Staphylococcus aureus dominant mucosal biofilms. However, S. aureus sinonasal mucosal colonisation occurs in the absence of inflammation, questioning the role of S. aureus in CRS pathogenesis. Here, we aimed to investigate the relationship between S. aureus biofilm metabolic activity and virulence genes, innate immune cells, and disease severity in CRS. Biospecimens, including sinonasal tissue and nasal swabs, and clinical datasets, including disease severity scores, were obtained from CRS patients and non-CRS controls. S. aureus isolates were grown into biofilms in vitro, characterised, and sequenced. The patients' innate immune response was evaluated using flow cytometry. S. aureus was isolated in 6/19 (31.58%) controls and 23/53 (43.40%) CRS patients of 72 recruited patients. We found increased S. aureus biofilm metabolic activity in relation to increased eosinophil cell frequencies and disease severity in recalcitrant CRS cases. Mast cell frequencies were higher in tissue samples of patients carrying S. aureus harbouring lukF.PV, sea, and fnbB genes. Patients with S. aureus harbouring lukF.PV and sdrE genes had more severe disease. This offers insights into the pathophysiology of CRS and could lead to the development of more targeted therapies.