Updated project metadata. The interaction between Aspergillus fumigatus (Af) spores and the respiratory epithelium, as well as its impact on the epithelial barrier function, remain largely unknown. The respiratory epithelial barrier protects the respiratory epithelium against viral infections. However, it can be compromised by environmental contaminants such as pollen, thereby increasing susceptibility to respiratory viral infections, including alphaherpesvirus equine herpesvirus type 1 (EHV-1). To determine whether Af spores disrupt the epithelial integrity and enhance susceptibility to viral infections, equine respiratory mucosal ex vivo explants were pretreated with Af spore diffusate, followed by EHV-1 inoculation. Af spore proteases were characterized by zymography and identified using mass spectrometry-based proteomics. Proteases of the serine protease, metalloprotease and aspartic protease groups were identified. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed that Af spores induced desquamation of epithelial cells and a significant increase in intercellular space at high and low concentrations, respectively. The increase in intercellular space in the epithelium caused by Af spore proteases correlated with an increase in EHV-1 infection. Together, our findings demonstrate that Af spore proteases disrupt epithelial integrity, potentially leading to increased viral infection of the respiratory epithelium.