Nanofibers prepared through the electrospinning process can be used in respiratory support devices and ventilators to produce filtering substrates. The size of the pores of these substrates should be adjusted based on the size of bacteria and viruses in order to provide the maximum possibility of air passing through the filter for the patient's breathing process. In this study, polyacrylonitrile nanofibrous substrates were produced using the industrial electrospinning process in optimized conditions of electrospinning with different voltage and speed settings. The performance of these substrates, including air permeability, water vapor permeability, filtration efficiency, pressure drop, and quality factor, was investigated using two samples of commercial substrates commonly used in respiratory support devices. To evaluate the efficiency of filtration, the designed device was used.The results showed that the maximum filtration efficiency of the produced nanofiber substrate against PM2.5 particles was 97.87% . The results indicated that samples produced at higher production speeds exhibited the highest Qf (>0.049 Pa⁻¹) and the lowest pressure drop (>39.24 Pa) at a high airflow rate of 60 L/min. Compared with commercial filters (QF ranging from 0.06 - 0.10 Pa⁻¹), the fabricated nanofiber samples demonstrated comparable or even superior performance. The obtained results showed that the produced substrates have suitable values for filtration and respiratory applications in terms of filtration performance, pressure drop, and quality factor.