This study investigated the influence of fiber content, temperature, and relative humidity on the thermal insulation properties of nonwoven mats made of seed fibers from Asclepias Syriaca, commonly known as milkweed floss. Nonwoven mats with a 1-inch thickness were produced by uniformly arranging milkweed fibers within a mold. Various quantities of fiber were employed to obtain nonwoven mats with a fiber content ranging from 5 to 35 kg/m3. Thermal conductivity and thermal diffusivity were measured across diverse relative humidity levels and temperatures. Simultaneously, milkweed floss samples were exposed to identical environmental conditions to assess the moisture regain and specific heat capacities of the fiber. The specific heat capacity of milkweed and thermal conductivity of the nonwovens exhibited a linear increase with temperature. The thermal diffusivity and thermal conductivity of the nonwovens decreased with rising fiber content. The thermal insulation properties of the nonwovens remained partially stable below 30% relative humidity but substantially deteriorated at higher levels. The nonwovens exhibited optimal thermal insulation properties at a fiber content between 20 and 25 kg/m3. The results of this study highlighted several technical advantages of employing milkweed floss as a sustainable and lightweight solution for thermal insulation.