Background: Topical surfactant therapy has been found to be effective in the management of recalcitrant chronic rhinosinusitis. Objective: To determine in vitro the antibacterial potential of SinuSurf™, a previously commercially available sinonasal surfactant. Methods: Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) cultures were grown in the presence or absence of serial dilutions of mupirocin or gentamicin with and without SinuSurf and quantified by colony-forming units (CFUs). Biofilm formation was also assessed. Finally, bacterial growth was evaluated in sinus irrigation bottles inoculated with MRSA or PA and rinsed daily with SinuSurf. Results: SinuSurf alone evinced a 3-log (1,000-fold) and 6-log (106-fold) reduction in CFUs for MRSA and PA, respectively. The combination of SinuSurf with a 1:10 dilution mupirocin and 1:100 dilution gentamicin demonstrated complete bacterial eradication. Similar concentrations of antibiotic dilutions alone demonstrated bacterial growth. SinuSurf averaged an 83% MRSA and 76% PA reduction in biofilm formation. Bottle contamination evaluation demonstrated reduction of MRSA and PA (p < 0.05) with SinuSurf. Conclusion: Biofilms have been demonstrated in chronic rhinosinusitis patients and implicated in recalcitrant disease. Our in vitro data demonstrates the addition of SinuSurf improved the effectiveness of a lower concentration of topical antibiotics in biofilm mass and viability.