Background: Staphylococcus aureus is the most common organism in recalcitrant chronic rhinosinusitis (CRS) and is often resistant to traditional antibiotic therapy. Bacteriophages ("phages") are a potential candidate for a new, effective therapy. For phages to be useful in the setting of CRS, two minimum requirements must be presented: (1) phages must be effective against S. aureus biofilms and (2) phages must have a broad spectrum of activity. This study aimed to assess the in vitro activity of a phage cocktail (CockTail of Staphylococcus aureus specific bacteriophage [CT-SA]) against S. aureus biofilms and a broad panel of strains isolated from patients with CRS. Methods: The study examined 66 clinical isolates (CIs) of S. aureus. All isolates were tested for the susceptibility to phage lysis by spotting CT-SA onto bacterial lawns. To measure its effect on S. aureus biofilms, a minimum biofilm eradication concentration assay was used, using five S. aureus isolates. Biofilms of these isolates were grown, treated with CT-SA for 48 hours, fluorescently stained, and viewed using confocal scanning laser microscopy. Results: CT-SA lysed 62 of 66 (94%) CIs of S. aureus. CT-SA treatment yielded significant reductions in biofilm mass for 4/5 CIs tested and for ATCC 25923. Challenge of S. aureus with a single phage resulted in the emergence of bacteriophage-insensitive mutants (BIM) with a frequency of 10-7, and challenge with CT-SA completely prevented their development. Conclusion: This study indicates that phage cocktail CT-SA can effectively eliminate S. aureus, in planktonic and biofilm forms, from the great majority of CIs from this hospital setting. In addition, its potential effect in preventing the emergence of BIMs was also established. Thus, CT-SA has the potential to treat S. aureus infection and biofilm in CRS patients.