Modern, intensive, high-density farming practices cause elevated concentrations of particulate matter (PM) inside livestock barns. PM in livestock barns is predominantly biological, hence, it contains abundant microorganisms. Understanding the microbial composition of PM is crucial for assessing the hazards of air emitted from livestock barns. PM and PM from a pig barn were collected in winter and spring, and morphological, chemical, and microbial analyses were performed. The PM samples exhibit diverse morphological characteristics. The top three elements detected in the PM samples were O, C, and Si. Other elements, including N, Al, K, Mg, Ca, Na, Zn, P, W, Ba, Fe, S, Cl, and Ti, were also identified in these samples. For bacterial α diversity, the Sobs and Chao1 indices for PM were significantly higher than those for PM in winter ( < 0.05), and in spring, the ACE index for PM was significantly higher than that for PM ( < 0.05). For fungal α diversity, the Shannon index for PM was significantly higher than that for PM in winter ( < 0.01), and in spring, the Ace index for PM was significantly higher than that for PM ( < 0.05). The β diversity results indicate that season, rather than the particle size, had a significant effect on the microbial composition in the PM samples. A total of seven bacterial pathogen genera and 16 fungal allergen genera were identified in PM samples. In winter, the relative abundances of total bacterial pathogens and fungal allergens in PM were higher than those in PM. In contrast, the relative abundance of fungal allergens in PM was higher in spring than in winter. This study provides a comprehensive characterization of PM from a pig barn across the particle sizes and seasons.