The paper deals with the cholesterol–cyclosporine A (Chol–CsA) monolayers at the air/water interface investigated using the Langmuir trough coupled with the Brewster’s angle microscopy. The compressed films were transferred onto the PEEK polymer support by means of the Langmuir–Blodgett technique. To improve molecules adhesion and organization the PEEK surface was treated with air plasma before thin films deposition. The obtained surfaces were characterized by means of atomic force microscope (AFM). Then, the wettability of the supported monolayers was determined by the contact angle measurements. Finally, the surface free energy and its components were evaluated from the theoretical approach proposed by van Oss et al. The obtained results reveal correlation between properties of the Langmuir monolayers at the air/water interface and those of the Langmuir–Blodgett films on PEEK. This was found to be helpful for understanding the wettability of organized molecular films on the polymer support as far as biocompatibility improve is concerned. The preparation of films with defined polarity and various compositions is an important step in the development of polymer surfaces with increased biofunctionality. It is believed that the results presented in this paper can be exploited in the in vivo studies.