Microorganisms are ubiquitous in the atmosphere, where they can disperse for a long distance. However, it remains poorly understood how these airborne microorganisms vary and which factors influence the microbial distribution in different anthropogenic activity regions. To explore the regional differences of bacteria and fungi in airborne particles, PM2.5 and PM10 samples were collected in the urban and rural areas of Hangzhou. The bacterial and fungal communities in the urban atmosphere was more similar to each other than those in the rural atmosphere. Analyses conducted by the concentration weighted trajectory model demonstrated that the local environment contributed more to the similarity of airborne bacteria and fungi compared with the atmospheric transport. The concentrations of local air pollutants (PM2.5, PM10, NO2, SO2 and CO) were positively correlated with the similarity of the bacterial and fungal communities. Additionally, the concentrations of these air pollutants in the urban site were about 1.5 times than those in the rural site. This implicated that anthropogenic activity, which is the essential cause of air pollutants, influenced the similarity of airborne bacteria and fungi in the urban area. This work ascertains the outdoor bacterial and fungal distribution in the urban and the rural atmosphere and provides a prospective model for studying the contributing factors of airborne bacteria and fungi. Display omitted •Airborne microorganisms were more similar in more urbanized site.•Atmospheric transport made contribution to the source of bacteria and fungi.•Local environment made a greater contribution to the source of bacteria and fungi.•Air pollutants were positively correlated with the similarity of bacteria and fungi. Exploring the distribution difference and influence factors of airborne bacteria and fungi in urban and rural areas shed light on the relationships between airborne microorganisms and air pollutants.