Particulate matter emissions from agricultural livestock operations contain both chemical and biological constituents that represent a potential human health hazard. The size and composition of these dusts, however, have not been well described. We evaluated the full size distribution (from 0 to 100 μm in aerodynamic diameter) and chemical/biological composition of inhalable dusts inside several Colorado dairy parlors. Four aerodynamic size fractions (<3, 3-10, 10-30, and >30 μm) were collected and analyzed using a combination of physiochemical techniques to understand the structure of bacterial communities and chemical constituents. Airborne particulate mass followed a bimodal size distribution (one mode at 3 μm and a second above 30 μm), which also correlated with the relative concentrations of the following microbiological markers: bacterial endotoxin, 3-hydroxy fatty acids, and muramic acid. Sequencing of the 16S-rRNA components of this aerosol revealed a microbiome derived predominantly from animal sources. Bacterial genera included Staphlyococcus, Pseudomonas, and Streptococcus, all of which have proinflammatory and pathogenic capacity. Our results suggest that the size distribution of bioaerosols emitted by dairy operations extends well above 10 μm in diameter and contains a diverse mixture of potentially hazardous constituents and opportunistic pathogens. These findings should inform the development of more effective emissions control strategies.