Wearing masks has become a new normal in our daily lives because of the global outbreak of COVID-2019 (COVID-19). To compare the differences in the facial microbiota of healthy women before and after wearing masks and to further explore the probable effect of the habits of regularly wearing masks on the facial microbiome, we re-enrolled the same 19 healthy female participants in our previous study and detected the microbial composition of facial DNA samples using 16S rRNA gene amplicon sequencing. Both alpha and beta diversity, and the abundance and function of facial microflora of recruited healthy women changed remarkably before and after wearing masks. The destination with different air quality indices, which ever was the strongest factor on microbial composition before wearing masks, no longer contributed to both microbiota composition and pathway after wearing masks. Sufficient sleep protected skin from sensitivity and apoptosis, which might be related to Prevotella expression and the function of cytochrome c. Maskne was the common complication of wearing masks. Suitable mask-wearing habits should be recommended to avoid facial skin problems.