AbstractObjectiveThis study aimed to expound on the correlation between facial skin microbiome and sensitive skin (SS) using a novel sequencing technique.MethodsWe applied the 2bRAD sequencing for the microbiome, which enables accurate characterization of the low‐biomass microbiome at species resolution to profile facial skin microbes in SS and non‐SS groups. Further, the bacterial colonies were isolated and cultured from skin surfaces to study the pro‐inflammatory effect on human keratinocytes by ELISA.ResultsWe accordingly identified 1142 genera and 4436 strains. In the SS group, the proportions of Actinomyces and Microbotryomycetes were significantly increased, whereas that of Acidimicrobiia was decreased. Kruskal–Wallis analysis revealed significant differences in 11 genera and 35 species, among which the proportions of Dermabacter, Chryseobacterium, Rhodotorula and Peptoniphilus A were increased in the SS group. Analysis of the top 10 genera revealed increased proportions of Cutibacterium, Corynebacterium and Staphylococcus. Moreover, the proportion of Dermabacter hominis was significantly increased by 18.9‐fold in the SS group, whereas those of many Streptococcus strains were significantly decreased. Focus on the isolated bacterial colonies from skin surfaces, more yellow colonies were found in SS group when cultured in Tryptic Soy Broth medium for 48 h, and more interleukin‐8 was detected on keratinocytes after yellow colonies stimulation, such as S.capitis, M.luteus.ConclusionsThis study suggests that more SS‐associated microorganisms can be identified using the 2bRAD technique even with a small sample size. Dermabacter hominis and Chryseobacterium was firstly reported with a significantly increase in SS, and the S.capitis, as well as M.luteus, but not S.aureus, may be associated with skin inflammation.