Hydrophobins are remarkable proteins due to their ability to self-assemble into amphipathic coatings that reverse surface wettability. Here, the versatility of the Class I hydrophobins EASΔ15 and DewY in diverse nanosuspension and coating applications is demonstrated. The hydrophobins are shown to coat or emulsify a range of substrates including oil, hydrophobic drugs, and nanodiamonds and alter their solution and surface behavior. Surprisingly, while the coatings confer new properties, only a subset is found to be resistant to hot detergent treatment, a feature previously thought to be characteristic of the functional amyloid form of Class I hydrophobins. These results demonstrate that substrate surface properties can influence the molecular structures and physiochemical properties of hydrophobin and possibly other functional amyloids. Functional amyloid assembly with different substrates and conditions may be analogous to the propagation of different polymorphs of disease-associated amyloid fibrils with distinct structures, stability, and clinical phenotypes. Given that amyloid formation is not required for Class I hydrophobins to serve diverse applications, our findings open up new opportunities for their use in applications requiring a range of chemical and physical properties. In hydrophobin nanotechnological applications where high stability of assemblies is required, simultaneous structural and functional characterization should be carried out. Finally, while results in this study pertain to synthetic substrates, they raise the possibility that at least some members of the pseudo-Class I and Class III hydrophobins, reported to form assemblies with noncanonical properties, may be Class I hydrophobins adopting alternative structures in response to environmental cues.