Background: Bacterial biofilms are thought to contribute to recalcitrance in chronic rhinosinusitis (CRS) patients. Manuka honey (MH) and its active component methylglyoxal (MGO) have demonstrated antibiofilm activity in vitro. This study evaluated the safety and efficacy of these agents in an in vivo model. Methods: To assess safety, ovine frontal sinuses were flushed twice daily for 14 days. In each sheep, 1 sinus was flushed with a panel of MGO concentrations ranging from 0.5 to 7.2 mg/mL alone and flushed with a panel of with 16.5% wt/vol MH enriched with MGO at the same range of concentrations (0.5-7.2 mg/mL; designated MH/MGO). Contralateral sinuses were flushed with saline control. Tissue morphology was assessed histologically and with scanning electron microscopy. Efficacy was tested by developing Staphylococcus aureus biofilms in sheep sinuses. Twice-daily irrigation for 5 days was commenced with either saline, MGO (0.5-3.6 mg/mL) alone, or MH/MGO (with 0.5-3.6 mg/mL MGO). Biofilm biomass was compared between the groups (n = 4) using LIVE/DEAD BacLight staining and confocal scanning laser microscopy. Results: The results of the safety assessment, for normal sinuses treated with MGO alone or with MH/MGO (≤1.8 mg/mL) showed normal pseudostratified epithelium and cilia structure; however, higher concentrations caused cilia denudation and squamous metaplasia. As for efficacy, when compared to saline flush, treatment with MH/MGO at 0.9 mg/mL (0.608 ± 0.110 vs 0.316 ± 0.197 μm3/μm2, respectively; p = 0.015) and 1.8 mg/mL (0.676 ± 0.079 vs 0.114 ± 0.033 μm3/μm2, respectively; p = 0.001) significantly reduced biofilm biomass. Conclusion: Sinus irrigation with MH/MGO at MGO concentrations between 0.9 and 1.8 mg/mL is both safe to mucosa and efficacious against S. aureus biofilm. MH/MGO irrigation could represent a viable treatment option for recalcitrant CRS.