Abstract Background Skin wound healing presents a complex challenge, often compounded by the risk of infection. Cold atmospheric plasma (CAP) emerged as a novel therapeutic for reducing bacterial load and expediting wound healing. However, its effect on the wound microbiome remained unclear. This study aimed to characterize the microbiome of different types of wounds and determine whether CAP influenced microbial diversity. Methods Twenty-five patients (ten with acute, fifteen with chronic skin wounds) and ten healthy controls were enrolled. CAP was tailored to individual clinical conditions. Skin samples were collected before and after CAP, and microbiota composition was determined by 16 S ribosomal RNA sequencing. Results Microbial communities differed between acute and chronic groups. CAP could accelerate wound healing. However, it did not change microbial α and β-diversity in acute wounds. In chronic wounds, α-diversity indices, including the chao and ACE, were significantly increased, and a significant clustering was observed in post-CAP group. In addition, CAP led to higher abundance of Staphylococcus, lower levels of Proteobacteria and Pseudomonas in chronic wounds. Conclusions This study provided novel insights into the impact of CAP on skin wound microbiota. Further research was required to ascertain causality between microbiota and CAP and to develop personalized CAP treatment strategies.