Objectives/Hypothesis Biofilms are associated with clinical relapse following surgery for chronic rhinosinusitis. Encased bacteria are protected from innate immunity and antimicrobial therapy. Surfactants can disperse the biofilm into its planktonic phenotype so that traditional treatments may be effective. The aim of this study was to assess a surfactant for its cytotoxicity profile. Study Design In vitro explant-based cytotoxicity study. Methods Sinonasal mucosa harvested from patients undergoing sinus surgery was tested using an air-liquid interface explant system. Surfactant at 1×, 2×, and 3× manufacturer's recommended concentrations were compared to control (saline) and Zinc Sulphate (ZnSO4), a known cytotoxic agent. Culture supernatant was analyzed for lactate dehydrogenase (LDH) as a marker of cellular toxicity. After 7 days, specimens were imaged using structured histopathology and scanning electron microscopy. Results Application of surfactant at 1× concentration did not elicit an elevation in LDH, whereas ZnSO4 caused a significant rise 1 day after application. Specimens tested with a 2× and 3× surfactant demonstrated LDH rises 4 days and 2 days after application, respectively. Mucosa tested with the 1× surfactant and control demonstrated intact cellular structures on histopathology and preserved cilial ultrastructure on SEM. In ZnSO4-treated specimens, marked cellular degradation and ciliary denudation occurred. Conclusion The surfactant does not appear to elicit cellular toxicity using an in vitro explant model at the manufacturer's recommended concentration. At higher concentrations, there may be dose-related toxicity that requires further investigation. In vivo testing is required to prove its efficacy in the treatment of recalcitrant chronic rhinosinusitis. Level of Evidence N/A.