Background:Seborrheic dermatitis (SD) is a common inflammatory skin condition. The etiology is unclear, although overgrowth of Malassezia on the skin has been suggested to cause SD. This study investigated whether colonization with Staphylococcus plays a role in facial SD, which was not well addressed previously.Methods:The study was conducted from September 1, 2011 to February 20, 2012 in the First Hospital of China Medical University. In the first phase, the study evaluated the level of transepidermal water loss (TEWL) and the number of colony-forming units (CFU) of Staphylococcus in defined skin areas of SD patients who were human immunodeficiency virus (HIV) seropositive (HIV [+] SD [+] group, n = 13), classical SD (HIV [−] SD [+] group, n = 24) patients, HIV seropositive-non-SD (HIV [+] SD [−] group, n = 16) patients, and healthy volunteers (HIV [−] SD [−] group, n = 16). In the second phase, we enrolled another cohort of HIV (−) SD (+) patients who applied topical fusidic acid (n = 15), tacrolimus (n = 16), or moisturizer (n = 12). Changes in the Seborrheic Dermatitis Area Severity Index (SDASI), TEWL, and Staphylococcus density were evaluated 2 weeks later. Comparisons of each index were performed using analysis of variance (ANOVA) and least significant difference method.Results:The level of TEWL was greater through lesional sites in the HIV (+) SD (+) group than that in HIV (+) SD (−) and HIV (−) SD (−) groups (95% confidence interval [CI]: 18.873–47.071, P < 0.001 and 95% CI: 28.755–55.936, P < 0.001, respectively). The number of CFU of Staphylococcus was greater in the HIV (+) SD (+) group than that in HIV (+) SD (−) and HIV (−) SD (−) groups (95% CI: 37.487–142.744, P = 0.001 and 95% CI: 54.936–156.400, P < 0.001, respectively). TEWL was significantly more improved in patients treated with tacrolimus and fusidic acid than that in those treated with moisturizers (95% CI: 7.560–38.987, P = 0.004 and 95% CI: 4.659–37.619, P = 0.011, respectively). Topical tacrolimus and fusidic acid were significantly associated with decreased SDASI as compared with moisturizer (95% CI: 0.03–0.432, P = 0.025 and 95% CI: 0.033–0.44, P = 0.024, respectively).Conclusions:High colonization with Staphylococcus epidermidis, along with impaired skin permeability barrier function, contributes to the occurrence of SD.