The interface between water and a hydrocarbonaceous material is important because of its usefulness for separation of a variety of chemical compounds. In order to change the magnitude of accumulation of the compounds at the water/hydrophobe interface, water miscible organic solvents such as acetonitrile are often added to the aqueous solution. However, the origin of the effect of the organic solvents on the separation of solute compounds has not been fully elucidated. We used surface-bubble-modulated liquid chromatography to investigate the spatial distribution of various organic compounds on an alkyl-bonded silica in contact with an acetonitrile–water mixture (0–5% (v/v)). Our results show that the dependences of the bulk liquid-to-aqueous/alkyl chain interface and bulk liquid-to-alkyl bonded layer distribution coefficients for organic compounds on acetonitrile concentration in aqueous solution are quite different from each other. It has been revealed that acetonitrile slightly partitions into the interior of the alkyl chain layer, whereas it preferentially accumulates at the interface although the interfacial liquid layer at the interface consists of not only acetonitrile but also water. The addition of acetonitrile to the aqueous solution causes a distinct decrease of accumulation of the compounds having hydrophilic groups, while the change in retention of hydrophobic compounds is rather small.