BACKGROUND: During the grass flowering season, fungal spores are abundant in outdoor air. We tested for co-sensitisations to grass pollen and fungal spores, assessed the degree of co-exposure, and studied its impact on the nasal mycobiome and immune responses. METHODS: Fungi-specific IgE-levels were studied in 277 individuals with and without grass pollen sensitisation. In a small cohort (n = 7), exposure to grass pollen and fungal spores was monitored during 5 consecutive indoor and outdoor stays in a flowering meadow and correlated with changes in the nasal mycobiome. Cytokines of nasal epithelial cells were studied under stimulation with recombinant grass pollen allergens, with and without fungal spores derived from outdoor isolates. RESULTS: IgE-sensitisation against the studied fungi was significantly more frequent among individuals with grass pollen sensitisation than among those without grass pollen sensitisation. Outdoor exposure resulted in changes in the nasal mycobiome, with a transitory enrichment of environmental fungi, for example, Cladosporium species. Most of the fungi cultivated from outdoor air samples belonged to the genera Fusarium, Cladosporium and Penicillium. Apical co-stimulation of nasal epithelial cells with grass pollen allergens and Fusarium, Cladosporium or Penicillium spores led to an increased loss of transepithelial electrical resistance and induction of pro-inflammatory cytokine release compared to mono-stimulation. CONCLUSION: Frequent co-exposure to fungal spores and grass pollen may increase the chance of acquiring a co-sensitisation to both allergens. Environmental fungi interact with and transitorily change the local mycobiome. Under co-exposure, fungal spores induce nasal inflammation and foster immune responses to otherwise poorly immunogenic pollen allergens.