Skin protects the body by mediating various immune responses against exogenous substances including bacteria, fungi, and viruses, in addition to its predominant role as a physical barrier. Despite the significant protection offered via various mechanisms, bacterial infection of the skin is one of the most common skin diseases in veterinary medicine. This study demonstrated the structural and immunological changes in the skin during infections with Pseudomonas aeruginosa and Staphylococcus pseudintermedius using skin explants from four healthy beagles. Skin structure was generally well preserved in uninfected controls, but defects in skin structure, including injury of keratinocytes and dermal–epidermal junctional disruption, were identified when skin explants were exposed to P. aeruginosa and S. pseudintermedius. On exposure to P. aeruginosa, marked linear cleft formation was noted along with acantholysis along the basal layer after 24 hours of culture. In addition to the defects in the skin structure, mRNA expression levels of the AMPs cBD103 and S100A8 were decreased, which was confirmed by immunohistochemical staining. Taken together, these results suggest that our ex vivo canine skin model is a research tool for investigating bacterial skin infections in dogs.