Microorganisms existing in airborne fine particulate matter (PM2.5) have key implications in biogeochemical cycling and human health. In this study, PM2.5 samples, collected in the typical basin cities of Xi’an and Linfen, China, were analyzed through high-throughput sequencing to understand microbial seasonal variation characteristics and ecological functions. For bacteria, the highest richness and diversity were identified in autumn. The bacterial phyla were dominated by Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Metabolism was the most abundant pathway, with the highest relative abundance found in autumn. Pathogenic bacteria (Pseudomonas, Acinetobacter, Serratia, and Delftia) were positively correlated with most disease-related pathways. Besides, C cycling dominated in spring and summer, while N cycling dominated in autumn and winter. The relative abundance of S cycling was highest during winter in Linfen. For fungi, the highest richness was found in summer. Basidiomycota and Ascomycota mainly constituted the fungal phyla. Moreover, temperature (T) and sulfur dioxide (SO2) in Xi’an, and T, SO2, and nitrogen dioxide (NO2) in Linfen were the key factors affecting microbial community structures, which were associated with different pollution characteristics in Xi’an and Linfen. Overall, these results provide an important reference for the research into airborne microbial seasonal variations, along with their ecological functions and health impacts.