Our previous study has shown that the self-assembly of several neurodegenerative-disease-related peptides in ambient water nanofilm condensed on mica is very sensitive to the amount of water on the surface. In this paper, we will demonstrate our hypothesis that the introduction of ethanol into the water nanofilm alters the properties of the interfacial water, resulting in changes of the peptide nanostructures self-assembled on the substrate. The assembly behaviors of peptides under different ethanol-containing atmospheres on mica were investigated by atomic force microscopy. GAV-9a began to form bent nanofilaments under an ethanol-containing atmosphere, and the self-assembled nanofilaments became thicker when a higher ratio of ethanol to water in the vapor was used. Based on these results, we propose a possible mechanism that the peptides adopt a "tilted upright" orientation when ethanol is present in the incubation environment. The effect of the peptide's terminal groups on the self-assembled nanostructures under the ethanol-containing atmosphere was also discussed.