Chlorhexidine (CHG) was found in perioperative surgical care. There have been no in vivo tests of CHG toxicity on human skin. Given the regular wound treatments and as a presurgical scrub, including donor site preparation, researchers wanted to determine whether CHG cytotoxicity would continue in a clinically relevant in vivo human skin xenograft model. Tissues from human skin were obtained through elective operations for research. A 4 mm punch biopsy was used to create partial-thickness wounds in human skin ex vivo.2% CHG (treatment) or PBS (control) was applied to the wounds for 30 minutes followed by rinsing the tissue +/- mechanical disruptive irrigation. Tissues were cultivated at the air-liquid interface for 24 hours in culture media after treatment, and tissue viability was assessed with an MTT assay. In vivo studies using athymic mice (n=4) were performed, in which human skin was grafted on both sides of their bilateral flanks. About 4 mm partial thickness wounds were created on each xenograft 8 weeks after engraftment and skin architecture normalization (2 in each mouse – treatment and control). About 2% CHG was applied daily for 2 minutes followed by irrigation with PBS in the treatment wound. The animal’s head was washed and then irrigated with a solution of peracetic acid. To simulate daily wound care, the xenografts were treated once a day for 14 days, and digital photographs were taken to document infection and substantial wound healing. On day 14, the xenografts were collected, stained for lactate dehydrogenase and H&E to assess cell viability and wound re-epithelization, and harvested. The author’s study on a mouse model of pressure ulcers showed that CHG treatment reduced the number of macrophages and neutrophils in the wound, but increased phagocytic activity. In the in vivo xenograft study, inflammation was not determined in either PBS or CHG-treated wounds at any point during the study. The wound size appeared to be greater on gross inspection in the CHG-treated group as early as day 2 compared with the PBS group. After 14 days of treatment, the PBS-treated wounds were completely re-epithelialized (n=2) or had substantially more re-epithelialization (n=2) microscopically than the CHG treated wounds (n=4). The irrigation procedure was not deadly to the cells, since the PBS-treated wounds were viable throughout the tissue. Nonviable cells were discovered in the dermis near to the wound, which was in touch with CHG, suggesting that penetration of CHG contributes to cytotoxicity in acute wounds.Daily CHG Use is cytotoxic to human skin and hinders wound healing.