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Dairy working increases the prevalence of lower airway respiratory diseases, especially COPD and asthma. Epidemiological studies have reported that chronic inhalation of organic dusts released during specific daily tasks could represent a major risk factor for development of these pathologies in dairy workers. Knowledge on size, nature and biological activity of such organic dusts remain however limited.To compare size distribution, microbial composition and cellular effects of dusts liberated by the spreading of straw bedding in five French dairy farms located in Brittany.Mechanized distribution of straw bedding generated a cloud of inhalable dusts in the five dairy farms’ barns. Thoracic particles having a 3–7.5 μm size constituted 58.9–68.3% of these dusts. Analyses of thoracic dusts by next generation sequencing showed that the microbial dust composition differed between the five French farms, although Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria represent more than 97.5% of the bacterial phyla detected in each sample. Several bacteria genera comprising of human pathogenic species, such as Pseudomonas, Staphylococcus, Thermoactinomyces or Saccharopolyspora were identified. Cladosporium and Alternaria fungal genera, which are potent environmental determinants of respiratory symptoms, were detected in dusts collected in the five farms and their levels reached 15.5–51.1% and 9–24.7% of assignable fungal sequences in each sample, respectively. Finally, all dust samples significantly and strongly increased the expression of the pro-inflammatory TNF-α, IL-1β, IL-6 and IL-8 cytokines at both mRNA and protein levels in human monocyte-derived macrophages. Their effects were dose-dependent and detectable from 1 μg/ml. The intensity of the macrophage responses however differed according to the samples.Our results strengthen the hypothesis that organic dusts released during the distribution of straw bedding are mainly constituted of thoracic particles which are small enough to deposit on lower bronchial epithelium of dairy farmers and induce inflammation.Distribution of straw bedding release a cloud of thoracic dusts.Thoracic dusts having a 3–7.5 μm size constitute the majority of inhalable dusts.Microbial composition of thoracic dusts has been analyzed by NGS.Thoracic dusts contain sequences related to bacterial and fungal pathogenic strains.Thoracic dusts induce a marked pro-inflammatory response of human macrophages.