AbstractBackgroundFungal involvement in asthma is associated with severe disease. The full spectrum of fungal species in asthma is not well described and is derived largely from insensitive culture techniques.ObjectivesTo use high‐throughput sequencing to describe the airway mycobiota in asthmatics with and without fungal sensitization and healthy controls; to compare samples representing different airway compartments; to determine whether the mycobiota was influenced by the fungal composition of outdoor air; and to compare findings with clinically relevant outcomes.MethodsWe amplified the internal transcribed spacer region 2 of the nuclear ribosomal operon to identify the fungal species present. Ninety‐seven subjects were recruited and provided sputum (83 asthmatics; 14 healthy subjects), with 29 also undergoing a bronchoscopy. A subset of airway samples were compared with matched outdoor air and mouthwash samples.ResultsTwo hundred and six taxa at the species level were identified in sputum, most at low relative abundance. Aspergillus fumigatus, Candida albicans and Mycosphaerella tassiana had the highest relative abundances and were the most prevalent species across all subjects. The airway mycobiota consisted of a complex community with high diversity between individuals. Notable shifts in the balance of fungi detected in the lung were associated with asthma status, asthma duration and biomarkers of inflammation. Aspergillus tubingensis, a member of the Aspergillus niger species complex, was most prevalent from bronchoscopic protected brush samples and significantly associated with a low sputum neutrophilia. Cryptococcus pseudolongus, from the Cryptococcus humicola species complex, was more abundant from bronchoscopy samples than sputum, and differentially more abundant in asthma than health.Conclusions and Clinical RelevanceThe airway mycobiota was dominated by a relatively small number of species, but was distinct from the oropharyngeal mycobiota and air samples. Members of the A. niger and C. humicola species complexes may play unexpected roles in the pathogenesis of asthma.