The two new p-tert-butylcalix[4]arene derivatives described here bear one or two nalidixic acid arms linked to the lower calixarene rim via the quinolone carboxylate moiety. These derivatives were synthesized in order to investigate two important features of molecules conceived as potential antibiotics, namely, metal cation complexation and interfacial properties, and the way in which they interrelate. The properties of the calixarene derivatives were studied in monomolecular films spread on pure water and on aqueous subphases containing biologically relevant mono- and divalent metal cations. These systems were examined via surface pressure and surface electrical potential measurements, polarization modulation infrared reflection absorption spectroscopy, and molecular modeling. Molecular modeling shows that important differences exist, first, between the structure and stability of the complexes formed with the two derivatives and, second, between their mono- and dication complexes. Correlating the properties of the monolayers with those of the modeled molecules lets us propose that the derivatives bearing one or two nalidixic pending arms form preferentially inter- and intramolecular complexes, respectively. The results obtained in this study indicate that a possible biological role of the nalidixic arms grafted on the calixarene crown may be revealed upon cation complexation.