The microbial composition of bioaerosols is an important issue to investigate to better understand occupational risks linked to exposure to airborne particles. Traditional methods are based on culture followed by colony identification by morphological, molecular biology or biochemical approaches. We have carried out a preliminary study which aims to generate reproducible complex bioaerosols and compares a high-throughput sequencing approach to the traditional culture-based approach. Mixed fungal bioaerosols containing four fungal strains (Aspergillus niger, Cladosporium cladosporioides, Penicillium brevicompactum and Wallemia melicolla) were generated in the laboratory using a bubbling aerosolizer. The fungal composition was assessed by culture and sequencing. Two genetic targets were sequenced and two assignment methods were compared. Wallemia was poorly detected by the culture-based approach, whereas Aspergillus was readily observed, in significant proportions, only by this method. The proportions in the liquid suspensions and in the bioaerosols generated were nearly identical. Sequencing of the ITS2 region provided results better reflecting the theoretical composition than 18S rDNA sequencing. The findings show that results of fungal composition investigations are strongly dependent on the analysis method used. Further studies will be required to assess the impact of these findings when considering biodiversity in complex and unknown environments.