A continuous high-throughput aerosol-based method for fabrication of antimicrobial air filters using natural antimicrobial nanoparticles was developed. We used the nebulization and electrospray methods for deposition of nanosized antimicrobial substances on pristine filter media. The roll-to-roll process was introduced for high-throughput fabrication of antimicrobial filters, and electrospray generation and dispersion equipment were used for high performance. The present method covers a filter area of 4500 mm2 at one time with uniform deposition. The characteristics of the airborne particles generated by nebulization and the electrospray method were evaluated using a scanning mobility particle analyzer (SMPS) and scanning electron microscopy (SEM). Furthermore, filter performance, such as the pressure drop and antimicrobial efficiency, was examined. The pressure drop of the antimicrobial filter showed a general increasing trend with amount of deposited antimicrobial particles. When 2.64 and 3.52 µg mm–2 of antimicrobial particles were loaded on pristine filter media, the measured antimicrobial efficiency of the filter was over 99.5% based on a 24-h contact time. This study provides useful information for the development of a high-throughput production process for antimicrobial air filtration systems.