The Langmuir technique allows for the characterization of monolayers of biological and/or biologically active compounds which have high potential for application in tissue engineering to improve the implants biocompatibility. This group of substances includes 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cyclosporine A (CsA), lauryl gallate (LG), and chitosan (Ch). The single (DOPC, CsA, LG), binary (DOPC-CsA 1:1) and ternary (DOPC-CsA-LG 3:3:2, 1:1:2, 1:1:6 that correspond to χLG = 0.25, 0.50, 0.75) monolayers on the acidic subphase with or without chitosan (Ch) were characterized using the Langmuir technique coupled with the surface potential meter. The dependence of the surface pressure and the surface potential on the area per molecule (π−A and ∆V−A isotherms, respectively) was determined. Based on the above isotherms data, the compression modulus (Cs−1), excess and total Gibbs energy of mixing (∆Gexc and ∆Gmix) as well as the apparent dipole moment (μa) were calculated. These parameters allowed drawing conclusions about the molecular packing, the orientation of molecules, the type and magnitude of interactions within the monolayers, and finally about their stability, depending on the components proportions and the contribution of Ch molecules. The main findings indicated that the insertion of LG to the binary DOPC-CsA 0.50 monolayer resulted in a shift of the π−A isotherms towards smaller areas per molecule, the smaller areas the higher amount of LG. That correlated with an increase in packing density (larger Cs−1 values) and an attraction between the molecules (revealed by the negative ∆Gexc and ∆Gmix values) in the three-component monolayers. Furthermore, the addition of chitosan (Ch) molecules into the subphase stiffened the multicomponent (DOPC-CsA or DOPC-CsA-LG) films. The strengthening of the attractive interactions and better miscibility were observed for all the DOPC-CsA-LG films. Therefore, these monolayers spread on the Ch subphase were found to be thermodynamically stable (among them the most stable was DOPC-CsA-LG 0.50) and could be successfully used for coating implants in order to improve their biocompatibility with the surrounding tissues.