Background During the COVID-19 pandemic, wearing PPE can induce skin damage such as erythema, pruritus, erosion, and ulceration among others. Although the skin microbiome is considered important for skin health, the change of the skin microbiome after wearing PPE remains unknown.Objective The present study aimed to characterize the diversity and structure of bacterial and fungal flora on skin surfaces of healthcare workers wearing personal protective equipment (PPE) during the COVID-19 pandemic using metagenomic next-generation sequencing (mNGS).Methods A total of 10 Chinese volunteers were recruited and the microbiome of their face, hand, and back were analysed before and after wearing PPE. Moreover, VISIA was used to analyse skin features. Results Results of alpha bacterial diversity showed that there was statistically significant decrease in alpha diversity indice in the skin samples from face, hand, and three sites after wearing PPE as compared with the indice in the skin samples before wearing PPE. Further, the results of evaluated alpha fungal diversity show that there was a statistically significant decrease in alpha diversity indices in the skin samples from hand after wearing PPE as compared with the indices in the skin samples before wearing PPE (P < 0.05). Results of the current study found that the main bacteria on the face, hand, and back skin samples before wearing the PPE were Propionibacterium spp. (34.04%), Corynebacterium spp. (13.12%), and Staphylococcus spp. (38.07%). The main bacteria found on the skin samples after wearing the PPE were Staphylococcus spp. (31.23%), Xanthomonas spp. (26.21%), and Cutibacterium spp. (42.59%). The fungal community composition was similar in three skin sites before and after wearing PPE. ConclusionIt was evident that wearing PPE may affect the skin microbiota, especially bacteria. Therefore, it was evident that the symbiotic microbiota may reflect the skin health of medical workers during the COVID-19 pandemic.