This study aims to investigate the influence of double‐layer clothing in a semiconductor manufacturing clean‐room environment. Twenty subjects including ten males and ten females participated in this study. Each subject completed four treatment combinations with four different inner pieces of clothing (i.e., 100% cotton, 70% cotton + 30% polyester, 65% polyester + 35% cotton, 100% polyester). The dependent measures included moisture absorption, skin temperature, and subjective responses in three body regions. The results indicate that wearing 100% polyester inner clothing caused a significant increase in inner microclimate relative humidity (RH; p < .01). Wearing 100% cotton inner clothing caused a significant increase in both inner microclimate RH (p < .01) and the inner clothing's moisture absorption. This was due to cotton fiber tends to trap more water molecules and allow less water vapor to pass through as compared to other types of fiber. Furthermore, wearing the blended fiber inner clothing caused lower RH in the inner and outer clothing microclimate (p < .05). Moreover, wearing 65% polyester + 35% cotton inner clothing had higher subjective comfort than did wearing 70% cotton + 30% polyester. In summary, the moisture absorption and water vapor transport characteristics of the inner clothing are the major factors affecting the comfort of wearing double‐layer clothing. © 2012 Wiley Periodicals, Inc.