ABSTRACTSkin microbiome dysbiosis with aStaphylococcusoverabundance is a feature of actinic keratosis (AK) and squamous skin carcinoma (SCC) patients. The impact of lesion-directed treatments for AK lesions such as diclofenac (DIC) and cold atmospheric plasma (CAP) on the lesional microbiome is not established. We studied 321 skin microbiome samples of 59 AK patients treated with DIC 3% gel versus CAP. Microbial DNA from skin swabs taken before start of treatment (week 0), at the end of the treatment period (week 24), and 3 months after end of treatment (week 36) was analyzed after sequencing the V3/V4 region of the16S rRNAgene. The relative abundance ofS. aureuswas scrutinized by atufgene specific TaqMan PCR assay. The total bacterial load and both, relative and absolute abundance ofStaphylococcusgenus was reduced upon both therapies at week 24 and 36 compared to week 0. Notably, the lesional microbiome of patients responding to CAP therapy at week 24 was characterized by an increased relative abundance ofCorynebacteriumgenus compared to nonresponders. A higher relative abundance ofStaphylococcus aureusat week 36 was a feature of patients classified as nonresponders for both treatments 12 weeks after therapy completion. The reduction of theStaphylococcusabundance after treatment of AK lesions and alterations linked to treatment response encourage further studies for investigation of the role of the skin microbiome for both, the carcinogenesis of epithelial skin cancer and its function as predictive therapeutic biomarker in AK.IMPORTANCEThe relevance of the skin microbiome for development of actinic keratosis (AK), its progression into squamous skin cancer, and for field-directed treatment response is unknown. An overabundance of staphylococci characterizes the skin microbiome of AK lesions. In this study, analyses of the lesional microbiome from 321 samples of 59 AK patients treated with diclophenac gel versus cold atmospheric plasma (CAP) revealed a reduced total bacterial load and reduced relative and absoluteStaphylococcusgenus abundance upon both treatments. A higher relativeCorynebacteriumabundance was a feature of patients classified as responders at the end of CAP-treatment period (week 24) compared with nonresponders and theStaphylococcus aureusabundance of patients classified as responders 3 months after treatment completion was significantly lower than in nonresponders. The alterations of the skin microbiome upon AK treatment encourage further investigations for establishing its role for carcinogenesis and its function as predictive biomarker in AK.