Adsorption to aquifer media is an important process in the removal of viruses from groundwater. Even though hydrophobic interactions have been shown to contribute to adsorption, little is known about the hydrophobicity of viruses found in groundwater. In this study, the hydrophobicity of rotavirus, MS2 bacteriophage and DNA-labelled silica nanoparticles (SiNPs) coated with glycoprotein, protein A and alpha-1-microglobulin/bikunin precursor (AMBP) was investigated. The hydrophobicity was experimentally determined by using a modified microbial adhesion to hydrocarbons (MATH) assay. The results were compared with the theoretical hydrophobicity of the viral capsid proteins and the proteins used to coat the nanoparticles, and with the results of adsorption tests with unmodified and organosilane-coated (hydrophobic) silica sand. While most theoretical protein hydrophobicity values were similar, the results of the MATH assay suggested fundamental differences in the hydrophobicity of the viruses and the SiNPs. MS2 was found to be highly hydrophobic as based on the MATH hydrophobicity and a significantly enhanced adsorption to hydrophobic sand, whereas rotavirus was relatively hydrophilic. The MATH assay revealed that protein-coating of SiNP introduced some degree of hydrophobicity to hydrophilic SiNPs, enabling them to more closely mimic viral hydrophobicity. Our study also demonstrated that the protein-coated SiNPs better mimicked rotavirus adsorption to sand media (coated or not coated with hydrophobic organic matter) than the MS2. This further supports previous findings that these surface-modified SiNPs are useful surrogates in mimicking rotavirus retention and transport in porous media.