Microbial aerosols in intensive broiler houses whose species and concentrations are closely related to human health are ubiquitous. Based on 16S rRNA gene sequencing, the aim of this study was to investigate the spatial distribution and diversity of bacterial aerosols in the air of broiler houses. Significant spatial variations in airborne bacterial concentrations were observed inside the poultry farmhouse. The results indicated that bacteria in the air samples could be grouped into a total of 1,674 OTUs. Alpha diversity analysis showed that the diversity of the microbial community at the entry of the broiler house was higher than that at the middle or the rear (p < 0.01). The Sankey diagram illustrated species dynamic changes in Proteobacteria, Firmicutes, and Actinobacteria among the different locations. From the aspect of LEfSe (LDA Effect Size) analysis, we discovered that the abundance of Planctomycetes was significantly higher in the entry than in the rear and middle. This study shows the spatial distribution of the entire bacterial community in intensive broiler houses, which offers a new perspective for studying airborne total bacteria in those environments.Implications: The bacteria contained in air aerosols from poultry houses are closely connected to animal health and production. This study aimed to investigate the spatial distribution and diversity of bacterial aerosols in the air of broiler houses. The results observed that bacterial aerosol concentrations in the examined broilers house varied greatly at different positions, and a significantly higher exposure to bacterial aerosol was observed at the middle than at the other positions (p < 0.05). The alpha diversity analysis showed that the diversity of the microbial community at the entry of the broiler house was higher than that at the middle or the rear (P<0.01). Sankey diagram illustrated species dynamic changes of Proteobacteria, Firmicutes and Actinobacteria among the different locations. The microbial communities in genus level in the samples of entry and rear were closer, while the species diversity of middle and rear samples in chicken house was highly similar (P>0.05). Altogether, results revealed that the effects of spatial factors on the diversity and abundance of bacteria in the air of closed-cage broiler houses, which poses a potential threat to the health of animals and workers in those environments.