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To assess indoor bioaerosols, a virtual impactor having 1 µm cutoff diameter was designed, fabricated, and evaluated with computational fluid dynamics simulation and also with laboratory test using polystyrene latex particles. Two other cutoff diameters of 635 nm and 1.5 µm were obtained by changing the inlet flow rate and the ratio of minor channel-to-inlet flow rates. In field test, the virtual impactor was operated with varying cutoff diameter and field-emission scanning electron microscope (FE-SEM) analysis was performed for each cutoff diameter to observe morphologies of indoor aerosol particles sampled at the major and minor outlet channels. Particles were sampled at both outlet channels using the SKC Button Aerosol sampler and subsequently cultured. By colony counting, it was found that 56% of cultured fungal particles and 63% of cultured bacterial particles had aerodynamic sizes smaller than 1 µm. MALDI-TOF analysis and visual inspection of culture samples were used to identify indoor bacterial and fungal species, respectively. Nearly same species of bacteria and fungi were detected both in the major and minor flow channels. © 2017 American Association for Aerosol Research [ABSTRACT FROM AUTHOR]