Wound infection is the most serious cause of delayed healing for patients with pressure injuries. The wound microbiota, which plays a crucial role in delayed healing, forms by bacterial dissemination from the peri-wound skin. To manage the bioburden, wound and peri-wound skin care has been implemented; however, how the microbiota at these sites contribute to delayed healing is unclear. Therefore, we investigated the relationship between healing status and microbial dissimilarity in wound and peri-wound skin.A prospective cohort study was conducted at a long-term care hospital. The outcome was healing status assessed using the DESIGN-R® tool, a wound assessment tool to monitor the wound healing process. Bacterial DNA was extracted from the wound and peri-wound swabs, and microbiota composition was analyzed using 16S rRNA gene analysis. To evaluate microbial similarity, the weighted UniFrac dissimilarity index between wound and peri-wound microbiota was calculated.Twenty-two pressure injuries (7 deep and 15 superficial wounds) were included in the study. For deep wounds, the predominant bacteria in wound and peri-wound skin were the same in the healing wounds, whereas they were different in all cases of hard-to-heal wounds. Analysis based on the weighted UniFrac dissimilarity index, there was no significant difference for healing wounds (p = 0.639), while a significant difference was found for hard-to-heal wounds (p = 0.047).Delayed healing is possibly associated with formation of wound microbiota that is different in composition from that of the skin commensal microbiota. This study provides a new perspective for assessing wound bioburden.