Mask-wearing behavior, common in the post-COVID-19 era, raises concerns for sensitive skin. This split-face study investigated mask-related changes in skin barrier function and microbiome composition among 30 female volunteers with sensitive skin and assessed the mitigating effects of a moisturizer containing biological lipids and probiotics. Skin physiological indicators (transepidermal water loss, erythema index, stratum corneum hydration, pH, temperature) of masked and unmasked areas were collected at baseline, after three hours of mask-wearing, post-tape stripping, and after 24 h, respectively. Microbiome samples collected from the masked areas before and after wearing a medical mask were analyzed with bioinformatics methods. Mask-wearing significantly weakened barrier function in both masked and adjacent unmasked areas, while reducing bacterial diversity. It was also associated with an increase in Cutibacterium (P = 0.110) and decreases in Streptococcus (P = 0.032) and Prevotella (P = 0.026) abundance. Moisturizer application prior to mask-wearing significantly reduced transepidermal water loss and erythema (both P < 0.001) and further improved erythema after 24 h (P = 0.048). These findings demonstrate that mask-wearing can disrupt the skin barrier and microbiome in individuals with sensitive skin and applying a moisturizer beforehand can mitigate mask-related discomforts by aiding barrier repair and reducing sensitivity.