Bacteria and fungi are primary constituents of airborne microbes in fine particulate matter and harmful to health. To evaluate the environmental quality of different poultry houses in Shandong Province, China, the airborne aerobic bacteria, airborne fungi, and airborne Escherichia coli were collected by the Andersen-6 air microorganism sampler. The fine particulate matter was collected by a ZR-3920 ambient air particulate matter sampler, and bacterial and fungal diversities and relative abundances analyzed using high-throughput sequencing. Results showed that the concentrations of airborne aerobic bacteria, airborne fungi, and airborne Escherichia coli in poultry houses were 0.167 to 4.484 × 104 CFU/m3, 0.236 to 4.735 × 103 CFU/m3, and 0 to 33.0 CFU/m3, respectively. 11.4 to 34.3% of aerobic bacteria and 16.8 to 37.5% of fungi were distributed at levels 5 and 6 (0.6 to 2.1 μm, the particle sizes similar to fine particulate matter) in the Andersen sampler. The concentration of fine particulate matter in the poultry houses was 114 to 230 μg/m3, which was higher than the safety value 10 specified by WHO. In fine particulate matter, the main bacteria at phylum level were Firmicutes, Bacteroidetes, and Proteobacteria, whereas the dominant phylum of fungus was Ascomycota and Basidiomycota. Importantly, the relative abundances of Escherichia and Corynebacterium in the broiler houses were greater than those in layer houses. However, the percentages of Aspergillus and Penicillium were 13.5 and 0.56%, with a relatively high level in the layer houses. Altogether, results revealed that the ambient air quality in the poultry houses sampled had a relatively high abundance of conditional pathogenic bacteria and concentration of fine particulate matter, which could threaten the health of animals and workers in those environments.