In recent years, the importance of using local disinfectants instead of systemic antibiotics for the treatment of infectious skin diseases to prevent the emergence of resistant bacteria has become widely recognized. Chlorhexidine gluconate (CHG) is commonly used in veterinary antibacterial shampoos
however, the daily topical application of diluted CHG solutions has also been adopted. Despite its widespread use, few studies have investigated the effects of CHG on the canine skin barrier. This study examined the skin barrier-damaging effects of CHG in dogs by applying diluted CHG (0.05%, 0.5%, and 4%) daily to six healthy dogs for 2 weeks. At the end of the trial, transepidermal water loss (TEWL), skin surface hydration (SSH), and number of skin bacteria were evaluated. Additionally, the cytotoxicity of CHG to the canine progenitor epidermal keratinocytes (CPEK) was examined using the water-soluble tetrazolium salt (WST-8) assay. Continuous application of 0.5% and 4% CHG to the skin led to a significant reduction in the number of skin bacteria. Additionally, at 4% CHG, a notable increase in TEWL and a decrease in SSH was observed. The WST assay revealed cytotoxicity of CHG at concentrations of 0.05%. In conclusion, although daily topical application of 4% CHG demonstrated the potential to disrupt the skin barrier, 0.5% CHG demonstrated sufficient antimicrobial activity without skin barrier disruption. Additionally, when treating dogs' skin with a compromised stratum corneum, use of lower concentration of CHG is suitable.