Orthopoxviruses, including smallpox, monkeypox and molluscipox, pose risks to human health through bioterrorist acts or natural transmission. There is no approved therapy for orthopoxvirus infections; however, cidofovir (CDV) has been approved as an investigational new drug for emergency treatment of adverse effects following smallpox vaccination. For evaluation of new therapies directed against orthopoxvirus infections, we have utilized immunocompetent, hairless mice (SKH-1) inoculated by a cutaneous route with cowpox virus (CV) or vaccinia virus (VV). Mice subsequently developed skin lesions and virus was recovered from the site of inoculation and quantified. Skin biopsies were evaluated microscopically, revealing brick-like eosinophilic, intracytoplasmic inclusion bodies characteristic of orthopoxvirus infection. SKH-1 mice fully recovered from either CV or VV infection. Immunodeficient Athymic or Rhino mice inoculated with CV or VV had more lesions and severe disease than SKH-1 mice. CV-infected SKH-1 mice were treated either with systemic or topical CDV. Although some protection was achieved with systemic treatment, 5% topical CDV was most effective at reducing virus titers in skin, lung, kidney, and spleen. These models may provide a means for evaluating efficacy of new therapies directed against orthopoxvirus diseases and further confirm the topical activity of CDV against cutaneous infections.