Clean room is an environment in which the concentration of ambient particles is controlled in order to keep the number of particles in the room at a minimum level. Clean room garments should be capable of preventing the transmission of particles from a person to the ambience. Filtration properties, along with favorable breathability and elasticity, provide a strong reason for using these structures for clothing purposes in the clean room environment. In the present study, the polyamide-66 nanofiber layer was electrospun on Spunbond-Meltblown-Spunbond nonwoven substrate as coating in several time intervals. Consequently, the filtration test was conducted on two groups of particles. Dioctyl phthalate as uniform particles and atmospheric particles as nonuniform particles were used to evaluate the filtration efficiency of the nanofibrous filter. The statistical analysis showed that the filtration efficiency for dioctyl phthalate particles was less than atmospheric particles. Also, at longer coating time, the difference between efficiencies was at the lowest level. Air permeability and water vapor permeability were studied as components of garment thermal comfort specifications. Results of this study show that air permeability decreased and overall filtration efficiency of the produced layer was improved by increasing of coating time.