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BACKGROUND: Biofilms represent a complex milieu of matrix-enclosed microorganisms, which can significantly contribute to the pathology of chronic wounds. In this study, we compare the activity of three commercial antimicrobial wound-care solutions, Vashe® (HOCl-based), PhaseOne® (HOCl-based), and Sulfamylon® (mafenide acetate), for their in vitro activity against bacterial and fungal biofilms. METHODS: Reference and clinical isolates of 6 Gram-negative bacterial species (36 total strains), 3 Gram-positive bacteria (21 strains), and 3 Candida species (9 strains) were used to create biofilms. Various working concentrations of the 3 antiseptic agents were incubated with the biofilms in microwell plates; they were monitored from 1 minute to 24 hours to compare bacterial and fungal viability through colony forming unit (CFU) analysis. RESULTS: Vashe® and PhaseOne® displayed excellent bactericidal and fungicidal activity, whereas Sulfamylon® demonstrated minimal activity against the biofilms tested. With the exception of C. albicans, all biofilms were eliminated at either 1 or 10 minutes using Vashe® and PhaseOne® solutions. In most cases, mafenide was unable to eliminate both bacterial and fungal biofilms, even with 24 hours of treatment. CONCLUSIONS: Biofilms represent a major clinical challenge, with no clear consensus for treatment of chronic wounds or prosthetic devices. Our results suggest that hypochlorous acid-based wound solutions such as Vashe® and PhaseOne® are more efficacious than mafenide in eliminating bacterial and fungal biofilms. Further studies are necessary to investigate and compare the in vivo efficacy of these products in clinical care.