Mold growth can trigger a variety of serious problems such as allergies and asthma. Designing surfaces that are unfavorable for the adhesion of fungal spores is considered an effective method to prevent fungal growth. In this study, the effect of hydrophilic surface treatment on the adhesion of fungal spores onto substrates was investigated using Aspergillus oryzae as a model fungus. The fungal spores that strongly adhered on the hydrophilic substrates under atmospheric conditions were easily removed by lightly washing by hand in water. These experimental results agreed well with thermodynamic predictions based on contact angle measurements. In addition, the removal ratio of the fungal spores on substrates coated with silica nanoparticles was higher than that on plasma-treated glass. It is believed that the contact area between a spore and substrate depended on the substrate roughness. Atomic force microscopy revealed that there was almost no adhesive force between the spores and glass substrate coated with silica nanoparticles. These results suggest that hydrophilic treatment using hydrophilic silica nanoparticles is more effective than hydrophilic plasma treatment to prevent fungal spore adhesion on glass substrates.