Cladosporium spp. and Alternaria spp. spores are dominating the airspora of Denmark. Currently, little is known about the influence of climate change on the fungal spore abundance in the air. The aim of this study was to examine temporal changes in airborne Alternaria and Cladosporium spores over 26 years. This is the first report of long-term airborne Cladosporium spore occurrence in Denmark. Air spore concentrations were obtained with a Burkard volumetric spore sampler placed in Copenhagen, Denmark, during June–September, 1990–2015. The highest monthly Spore integrals (SIn) for Alternaria were measured in August, whereas for Cladosporium July SIn was nearly as high as August SIn. Average Alternaria seasonal spore integral (SSIn) was 8615 Spores day m−3, while average 3-month (July–September) Cladosporium SIn was 375,533 Spores day m−3. Despite increasing annual temperature and decreasing relative humidity, we found a decreasing trend for Alternaria seasonal SIn (Slope = − 277, R2 = 0.38, p < 0.05), Alternaria (Slope = − 31, R2 = 0.27, p < 0.05) and Cladosporium (Slope = − 440, R2 = 0.23, p < 0.05) annual peak concentrations. We did not find any statistically significant trends for airborne Alternaria seasonal characteristics and duration, and likewise for Cladosporium 3-month SIn and peak concentration dates. Mean temperature was the main meteorological factor affecting daily spore concentrations. However, effect of meteorological parameters on daily spore concentrations was stronger for Cladosporium (R2 = 0.41) than for Alternaria (R2 = 0.21). Both genera had diurnal peaks during the day hours, earlier for Cladosporium (11:30–14:30) and later for Alternaria (15:00–19:00). Although Alternaria and Cladosporium daily concentrations were moderately correlated (Spearman’s correlation coefficient: rs = 0.55, p < 0.05), their overall annual indices were different, which indicates different sources and different factors determining spore release. We explain temporal decreasing trends in Alternaria SSIn by growing urbanisation around Copenhagen and by changes in agricultural practices.