Actinic keratosis (AK) occurs on sun-damaged skin and is considered a precursor to squamous cell carcinoma (SCC). Photodynamic therapy (PDT), using 5-aminolevulinic acid (ALA) and red light, is a common treatment for AK. However, its clinical efficacy for invasive tumors such as SCC is limited by the poor penetration and distribution of the photosensitizer. Cold atmospheric plasma (CAP), a partially ionized gas, increases skin permeability and exhibits anti-cancer properties through the generation of reactive oxygen species (ROS). In a previous study, CAP showed promising synergistic effects when combined with ALA-PDT for the treatment of SCC cells in vitro. The present study investigated the effects of combining CAP with ALA-PDT on cutaneous AK and SCC induced by ultraviolet B (UV-B) irradiation in SKH1 hairless mice. We compared various application sequences (CAP-ALA-red light, ALA-red light-CAP, and ALA-CAP-red light) against conventional ALA-PDT using visual, histological, and molecular assessments of the affected skin. The results demonstrated that combined treatments strongly inhibited the growth of UV-B-induced skin lesions. TUNEL staining revealed increased apoptosis following both single and combined therapies, while Ki-67 staining indicated reduced keratinocyte proliferation and diminished DNA damage in treated areas. mRNA expression analysis showed the upregulation of apoptosis-related genes (, ) alongside enhanced anti-tumor immune responses (, ) in the affected tissue samples. Notably, the combined treatment enhances the therapeutic effect, whereas the sequence of application does not seem to be relevant for therapeutic efficacy in vivo. Overall, these results suggest that CAP may enhance the anti-tumor effect of conventional ALA-PDT, supporting previous findings on SCC cells.