Measurements of ambient aerosol particles at the University of León, León, Spain, were made in May and June 2015 with a Wideband Integrated Bioaerosol Spectrometer (WIBS). The WIBS detects Fluorescing Aerosol Particles (FAP) in the size range from 0.5 to 20 μm. These measurements were complemented with an analysis of pollen concentrations assessed with optical microscopy of samples captured with a volumetric Hirst spore trap. The total particle, FAP and pollen concentrations show clear, daily cycles. Whereas the total particle concentrations maximize at 0800 and 2200 UTC, the FAP concentrations have peaks at midnight and 0800 UTC while the pollen has a broad peak between 1200 and 2000 UTC. The FAP larger than 2 μm represent 15–35% of the total particle population in this size range, maximizing at midnight UTC. Similar to what has been found by investigators at other locations, there is a strong positive correlation of the WIBS measured FAP with relative humidity; however, the pollen concentration is positively correlated with the temperature and anti-correlated with the relative humidity. Back trajectory analysis indicates that the largest FAP to total particle fractions are found in air masses arriving from the northeast with the second largest coming from the southwest. Given the location of the university in relation to the city and forested areas, this implies that the higher concentration FAP are coming from rural, probably natural, sources; however, more local, anthropogenic sources cannot be ruled out as a secondary source. The majority of the FAP that are identified from microscopy are fungal spores (Cladosporium, Aspergillus, Alternaria, Oidium) and pollen grains (mainly Poaceae, Quercus, Plantago, Rumex and Urticaceae). A comparison of the fluorescence fingerprints between laboratory generated FAP and the ambient particles showed some similarities; however, a significant fraction of the FAP are those whose fluorescence patterns do not match any of those that have been previously classified in the laboratory. •A WIBS and a Hirst-type volumetric trap measured bioaerosols simultaneously in Leon.•The bioaerosol sizes determined by the WIBS are consistent with microscope images.•The WIBS Type III class and Hirst pollen concentration were of comparable magnitude.•Use of the WIBS and biolibrary is a potential approach for pollen classification.•The WIBS could become a useful tool for pollen quantification in real time.