Povidone-iodine (PVP-I) is an antiseptic and a disinfectant with broad-spectrum antimicrobial activity against various pathogens. However, it is unclear whether PVP-I nasal instillation can suppress mucosal inflammation in non-eosinophilic chronic rhinosinusitis (CRS) mice. This study aimed to explore the anti-inflammatory effects and underlying molecular mechanism of PVP-I on lipopolysaccharide-stimulated airway epithelial cells and investigate whether nasal instillation of PVP-I can suppress mucosal inflammation in non-eosinophilic CRS mice. Inflammation-related molecules in the nasal epithelial cells and non-eosinophilic CRS mice were measured by enzyme-linked immunosorbent assay, western blotting, quantitative real-time polymerase chain reaction, immunoprecipitation, and histopathological analysis. PVP-I blocked expressions of various inflammation-related molecules, such as NLRP3, NF-κB-p65, caspase-1, and IL-1β. Translocation of NF-κB to the nucleus, and assembly of NLRP3/ASC complexes in the nasal epithelial cells and non-eosinophilic CRS mice were also restricted. Notably, PVP-I strongly blocked the receptor co-localization of TLR4 and MyD88 in the epithelial cells of nasal mucosa. We demonstrated that PVP-I significantly attenuated inflammatory molecules and cytokines via blocking the formation of TLR4 and MyD88 complexes during LPS-induced mucosal inflammation in non-eosinophilic CRS.