A total of 480 microorganisms collected from 39 clouds sampled at the Puy de Dôme station (alt. 1465 m; 45°46′19′′ N, 2°57′52′′ E; Massif Central, France) were isolated and identified. This unique collection was screened for biosurfactant (surfactants of microbial origin) production by measuring the surface tension (σ) of the crude extracts, comprising the supernatants of the pure cultures, using the pendant drop technique. The results showed that 41 % of the tested strains were active producers (σ < 55 mN m−1), with 7 % being extremely active (σ < 30 mN m−1). The most efficient biosurfactant producers (σ < 45 mN m−1) belong to a few bacterial genera (Pseudomonas and Xanthomonas) from the Υ-Proteobacteria class (78 %) and a yeast genus (Udeniomyces) from the Basidiomycota phylum (11 %). Some Bacillus strains from the Firmicutes phylum were also active but represented a small fraction of the collected population. Strains from the Actinobacteria phylum in the collection examined in the present study showed moderate biosurfactant production (45<σ < 55 mN m−1). Pseudomonas (Υ-Proteobacteria), the most frequently detected genus in clouds, with some species issued from the phyllosphere, was the dominant group for the production of biosurfactants. We observed some correlations between the chemical composition of cloud water and the presence of biosurfactant-producing microorganisms, suggesting the “biogeography” of this production. Moreover, the potential impact of the production of biosurfactants by cloud microorganisms on atmospheric processes is discussed.