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 (P<0.05), with the highest levels (GM: dust, 1.38 mg/m(3); endotoxin, 895 EU/m(3); β(1 → 3)-glucan, 7.84 μg/m(3)) in barns with compost bedding vs the lowest in barns with sawdust bedding (GM: dust, 0.51 mg/m(3); endotoxin, 183 EU/m(3); β(1 → 3)-glucan, 1.11 μg/m(3)). The exposure levels were also highly variable, depending on various extra bedding materials applied. Plant materials, particularly straw, utilized for bedding appeared to be a significant source for β(1 → 3)-glucan. Compost was significantly associated with elevated exposure levels. Between-worker variances of exposure were highly explained by determinants of exposure like type of bedding materials and milking by robot, whereas determinants could explain to lesser extent the within-worker variances. Exposure levels to endotoxin, β(1 → 3)-glucan, bacteria, and fungi in dairy barns were substantial and differed depending on bedding materials, suggesting bedding material types as a significant predictor of bio-aerosol exposure.