Background: Saline-based irrigation solutions are evidence-based rhinological treatments; however, the formulation of these solutions could theoretically alter the function of innate antimicrobial peptides. The aim of this study was to determine if the antimicrobial activity of normal human nasal secretions in vivo is altered by commercially available large volume irrigation solutions. Methods: Minimally manipulated sinonasal secretions were collected from patients with chronic rhinosinusitis (CRS; n = 10) and normal healthy volunteers (n = 20). In a subset of control patients (n = 10) secretions were collected prior to, and at 1 hour, 6 hours, and 24 hours after nasal irrigation with 4 commercial irrigation solutions. Lysozyme and lactoferrin levels were analyzed and the antimicrobial activity of secretions determined using a radial diffusion assay. Results: The antimicrobial activity of nasal secretions was reduced in CRS patients compared to healthy volunteers (p < 0.01), but there was no significant difference in antimicrobial peptide concentrations. Isotonic nasal irrigation reduced lysozyme and lactoferrin levels, which returned to baseline levels by 6 hours; in addition to a sustained decrease in antimicrobial activity before returning to baseline at 24 hours. Low-salt solution stimulated peptide secretion by approximately 40% at 6 hours and 24 hours, but produced a transient decrease in antimicrobial activity, returning to baseline levels by 6 hours. Hypertonic solution initially decreased lysozyme and lactoferrin levels but maintained baseline levels of antimicrobial activity and increased peptide secretion by approximately 30% at 24 hours. Conclusion: The formulation of nasal irrigation solutions significantly affects the measured levels and functionality of sinonasal antimicrobial peptides.