Work in animal production facilities often results in exposure to organic dusts. Previous studies have documented decreases in pulmonary function and lung inflammation among workers exposed to organic dust in the poultry industry. Bacteria and fungi have been reported as components of the organic dust produced in poultry facilities. To date, little is known about the diversity and concentration of bacteria and fungi inside poultry buildings. All previous investigations have utilized culture-based methods for analysis that identify only biota cultured on selected media. The bacterial tag-encoded flexible (FLX) amplicon pyrosequencing (bTEFAP) and fungal tag-encoded flexible (FLX) amplicon pyrosequencing (fTEFAP) are modern and comprehensive approaches for determining biodiversity of microorganisms and have not previously been used to provide characterization of exposure to microorganisms in an occupational environment. This article illustrates the potential application of this novel technique in occupational exposure assessment as well as other settings. An 8-hr area sample was collected using an Institute of Medicine inhalable sampler attached to a mannequin in a poultry confinement building. The sample was analyzed using bTEFAP and fTEFAP. Of the bacteria and fungi detected, 116 and 39 genera were identified, respectively. Among bacteria, Staphylococcus cohnii was present in the highest proportion (23%). The total inhalable bacteria concentration was estimated to be 7503 cells/m³. Among the fungi identified, Sagenomella sclerotialis was present in the highest proportion (37%). Aspergillus ochraceus and Penicillium janthinellum were also present in high proportions. The total inhalable fungi concentration was estimated to be 1810 cells/m³. These estimates are lower than what has been reported by others using standard epifluorescence microscope methods. However, no study has used non-culture-based techniques, such as bTEFAP and fTEFAP, to evaluate bacteria and fungi in the inhalable fraction of a bioaerosol in a broiler production environment. Furthermore, the impact of this bTEFAP and fTEFAP technology has yet to be realized by the scientific community dedicated to evaluating occupational and environmental bioaerosol exposure.