Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by TH2-skewed inflammation and increased colonization by Staphylococcus aureus. IFN-lambda 1 is known for its antiviral activity, but there is little information on its antibacterial role. Objective: We sought to determine the expression and release of IFN-lambda 1 from nasal mucosal tissue of healthy subjects and patients with CRSwNP on exposure to S aureus and assess its potential role in antibacterial defense mechanisms. Methods: Nasal tissue from healthy subjects and patients with CRSwNP was exposed to S aureus, and we assessed expression of IFN-lambda 1, MUC5AC, and MUC5B. THP1-derived macrophages incubated with or without IFN-lambda 1 were assessed for uptake and killing of S aureus and expression of lysosomal-associated membrane protein 1 and intracellular reactive oxidase substrate (ROS), the IFN-lambda 1 receptor IL-28 receptor (IL-28R), and the Janus kinase/signal transducer and activator of transcription (STAT) 1 pathway by means of immunofluorescence staining. Results: S aureus infection increased IFN-lambda 1 expression in tissue from patients with CRSwNP. IFN-lambda 1 (10 ng/mL) significantly decreased the number of S aureus colony-forming units in healthy control tissue but not in tissue from patients with CRSwNP and upregulated MUC5AC and MUC5B expression in control tissue on S aureus infection. IFN-lambda 1 stimulation increased intracellular killing of S aureus in THP1-derived macrophages and substantially increased lysosomal-associated membrane protein 1, IL-28R, ROS, and STAT signaling in macrophages incubated with S aureus. All of these effects were attenuated by blocking IL-28R and ROS activities. Conclusions: IFN-lambda 1 favors clearance of S aureus in healthy nasal mucosa and enhances antibacterial function of macrophages through IFN-lambda 1-IL-28R-ROS-Janus kinase-STAT signaling pathways.