Mixtures of azo-functionalized amphiphilic derivatives of guanosine and of amphiphilic derivatives of other DNA nucleobases were deposited at an air-water interface and repeatedly irradiated with light of 340 and 440 nm wavelengths. The consequent switching between cis and trans configurations of the azobenzene moiety caused changes in the surface pressure of the film, which were analyzed using a model based on the two-dimensional Van der Waals equation of state. For mixed films of guanosine and cytidine derivatives, the analysis revealed a significant modification of the strength of intermolecular interaction caused by the optical irradiation, while no such modifications were identified in mixed films involving other nucleobases. The difference is attributed to light-induced breaking of the hydrogen bonding that is established only between specific nucleobases. The results demonstrate that photosensitive nucleoside derivatives can be used as an efficient probe for base-pairing in Langmuir monolayers.