Cadexomer iodine releases iodine (0.9% weight/weight) slowly from beads of dextrin and epichlorhydrin. This preparation is an effective debridement and antiseptic agent for chronic exdudative wounds. The purpose of the present study is to examine the influence of cadexomer iodine against glycocalyx production of Staphylococcus aureus isolated from furuncle lesions on cut wounds in mice using confocal laser scanning microscope (CLSM), and the increase in and glycocalyx production of S. aureus in vitro. In the present study, distinct S. aureus cells and glycocalyx were not detected in the dermis around the cadexomer iodine beads or within those beads, while S. aureus cells encircled by glycocalyx were soaked up by the cadexomer beads and were detected within them in vivo and in vitro. We suggest that cadexomer iodine soaks up S. aureus cells encircled by glycocalyx, directly destroys biofilm structures, and collapses glycocalyx during dehydration, and further, that iodine can subsequently kill S. aureus cells within biofilm. Cadexomer iodine is a promising treatment to clear S. aureus cells within biofilm from skin lesions of exudative or infectious wounds and to prevent wound exacerbation.