Summary Background Personal exposure to fungal bioaerosols derived from contaminated building materials or agricultural commodities may induce or exacerbate a variety of adverse health effects. The genomic mechanisms that underlie pulmonary immune responses to fungal bioaerosols have remained unclear. Objective The impact of fungal viability on the pulmonary microRNA and messenger RNA profiles that regulate murine immune responses was evaluated following subchronic inhalation exposure to Aspergillus fumigatus conidia. Methods Three groups of naïve B6C3F1/N mice were exposed via nose‐only inhalation to A. fumigatus viable conidia, heat‐inactivated conidia (HIC), or HEPA‐filtered air twice a week for 13 weeks. Total RNA was isolated from whole lung 24 and 48 h postfinal exposure and was further processed for gene expression and microRNA array analysis. The molecular network pathways between viable and HIC groups were evaluated. Results Comparison of data sets revealed increased Il4, Il13 and Il33 expression in mice exposed to viable vs. HIC. Of 415 microRNAs detected, approximately 50% were altered in mice exposed to viable vs. HIC 48 h postexposure. Significantly down‐regulated (P ≤ 0.05) miR‐29a‐3p was predicted to regulate TGF‐β3 and Clec7a, genes involved in innate responses to viable A. fumigatus. Also significantly down‐regulated (P ≤ 0.05), miR‐23b‐3p regulates genes involved in pulmonary IL‐13 and IL‐33 responses and SMAD2, downstream of TGF‐β signalling. Using Ingenuity Pathway Analysis, a novel interaction was identified between viable conidia and SMAD2/3. Conclusions and Clinical Relevance Examination of the pulmonary genetic profiles revealed differentially expressed genes and microRNAs following subchronic inhalation exposure to A. fumigatus. MicroRNAs regulating genes involved in the pulmonary immune responses were those with the greatest fold change. Specifically, germinating A. fumigatus conidia were associated with Clec7a and were predicted to interact with Il13 and Il33. Furthermore, altered microRNAs may serve as potential biomarkers to evaluate fungal exposure.