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From previous studies, we have shown that viable colony forming units of bacteria and bacterial biofilms are reduced after sequential treatment with a surfactant‐based dressing. Here, we sought to test the impact on visible bacterial pigments and the ultrastructural impact following the sequential treatment of the same surfactant‐based dressing. Mature Pseudomonas aeruginosa biofilms were grown on ex vivo porcine skin explants, and an imaging‐based analysis was used to compare the skin with and without a concentrated surfactant. In explants naturally tinted by bacterial chromophores, wiping alone had no effect, while the use of a surfactant‐based dressing reduced coloration. Similarly, daily wiping led to increased immunohistochemical staining for P. aeruginosa antigens, but not in the surfactant group. Confocal immunofluorescent imaging revealed limited bacterial penetration and coating of the dermis and loose pieces of sloughing material. Ultrastructural analysis confirmed that the biofilms were masking the extracellular matrix (ECM), but the surfactant could remove them, re‐exposing the ECM. The masking of the ECM may provide another non‐inflammatory explanation for delayed healing, as the ECM is no longer accessible for wound cell locomotion. The use of a poloxamer‐based surfactant appears to be an effective way to remove bacterial chromophores and the biofilm coating the ECM fibres. [ABSTRACT FROM AUTHOR]