Thermal protective clothings are produced from multilayered textile materials. Fabric layers need to allow enough evaporation of perspiration, ventilation, and also thermal protection from fire. This study aimed to evaluate the effects of different fabric layers and their different combinations on the thermal properties of multilayered fabric samples. Three-layered fabric combinations were created using two types of outer shell fabrics, four types of moisture barrier fabrics with membrane, and two types of thermal barrier fabrics. Sixteen different fabric combinations that simulate three-layered thermal protective clothing were studied. As a result of the study, it was found that thermal and moisture comfort properties were significantly affected by different fabric layers.