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Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins–hydrophobins (HFBs)–that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness. Author summary: Hydrophobins (HFBs) are unique, non-toxic small fungal proteins with outstandingly high surface activity. They modulate surface properties by self-assembling in interfaces and have a multitude of applications for drug delivery, biosensors, and cosmetics. To explore the applied potential of HFBs, we investigated their functions in the naturally HFB-enriched Trichoderma spp. fungi (Ascomycota). We show that HFBs unexpectedly specifically accumulate inside aerial hyphae, where they associate with lipid-enriched organelles and putatively line up the vacuolar structures, which contribute to the structure and longevity of aerial mycelium. The maximum of HFB synthesis and secretion happens before the sporulation, providing a massive extracellular matrix suitable for the protective coating of rapidly produced spores. Furthermore, HFBs are involved in the water sensing mechanism of spores and thus are linked to the dormancy/germination switch. Our results reveal that HFBs have a broad range of intracellular functions and are essential for fungal development and fitness. [ABSTRACT FROM AUTHOR]