Bio-aerosol is a well-known cause of respiratory diseases. Exposure to bio-aerosols has been reported previously in dairy barns, but little is known about the sources of bio-aerosol. Bedding materials might be a significant source or substrate for bio-aerosol exposure. The aim of this study was to explore bio-aerosol exposure levels and its determinants in dairy barns with various bedding materials. Dust samples were collected at dairy barns using various bedding materials. Samples were analyzed for endotoxin and β(1 → 3)-glucan contents. Culturable bacteria and fungi were sampled by the Anderson N6 impactor. Exposure models were constructed using linear mixed models. The personal exposure levels to dust, endotoxin, and β(1 → 3)-glucan differed significantly between the barns utilizing diverse main bedding types (