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Previously, we showed that chitosan could augment the biocidal efficacy mediated by photodynamic treatment against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. In this study, we showed that the antimicrobial action of chitosan in augmenting photodynamic inactivation (PDI) is related to the increase in cell surface destruction. The microbial cell surfaces exhibit severe irregular shapes after PDI in the presence of chitosan as demonstrated by transmitted electron microscopy. Furthermore, increases in the concentration or incubation time of chitosan significantly reduced the amounts of photosensitizer toluidine blue O required, indicating that chitosan could be an augmenting agent used in conjunction with PDI against S. aureus, P. aeruginosa, and C. albicans. A prolonged lag phase was found in microbial cells that survived to PDI, in which chitosan acted to completely eradicate the cells. Once the exponential log stage and cell rebuild began, their cellular functions from PDI-induced damage returned and the increased cytotoxic effect of chitosan disappeared. Together, our results suggest that chitosan can prevent the rehabilitation of PDI-surviving microbial cells, leading to increased biocidal efficacy.