Background: Prolonged exposure to environmental antigens or allergens elicits an immune response in both healthy horses and those with mild asthma. Corticosteroids often are used to treat lower airway inflammation.Objective: To investigate the changes in equine herpesvirus (EHV)-1,2,4,5 glycoprotein B gene expression and changes in respiratory bacterial and fungal communities after nebulized dexamethasone treatment of horses with asthma.Animals: Horses with naturally occurring mild asthma (n = 16) and healthy control horses (n = 4).Methods: Prospective, randomized, controlled, blinded clinical trial. Polymerase chain reaction amplification of EHV-1,2,4,5 in bronchoalveolar lavage fluid, and 16S (microbiome) and ITS2 (mycobiome) genes with subsequent sequencing was performed on DNA extracted from nasal swabs and transendoscopic tracheal aspirates before and after 13 days treatment with nebulized dexamethasone (15 mg q24h) and saline (control).Results: Nebulized dexamethasone treatment decreased microbial diversity; relative abundance of 8 genera in the upper respiratory tract were altered. For both the microbiota and the mycobiota, environment had a dominant effect over treatment.Alternaria, an opportunistic pathogen and allergen in humans recognized as a risk factor for asthma, asthma severity, and exacerbations, was increased with treatment.Treatment affected relative quantification of the equine gamma herpesviruses (EHV-2 and -5); EHV-2 DNA levels increased and those of EHV-5 decreased.Conclusions: Nebulized dexamethasone treatment affected the upper respiratory tract microbiota, but not the mycobiota, which was overwhelmed by the effect of a sustained dusty environment. K E Y W O R D S equine herpesvirus, mild equine asthma, qPCR, respirable dust, REST analysis Abbreviations: EHV, equine herpesvirus; BALF, bronchoalveolar lavage fluid; OTU, operational taxonomic unit; PCoA, principal coordinates analysis.