The wetting properties of chitosan (Ch) and single 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and binary DPPC-Chol layers deposited onto polyethylene terephthalate activated by low-temperature air plasma (PETair) were examined. PET is widely used in tissue engineering, but its low hydrophilicity limits its integration with the surrounding tissues. Ch is a biocompatible polysaccharide, distinguished by its antimicrobial properties, widely distributed in medicine. DPPC and Chol are the major building components of cell membrane, so they can perfectly mimic membrane behaviour during contact with the Ch layer. Monolayers of lipids were deposited onto PETair with or without the Ch layer using the Langmuir-Blodgett technique. The total surface free energy (SFE) and its components changes were calculated from theoretical approaches. Wettability strongly depended on the monolayer composition as well as the Ch layer. The Ch film decreased the contact angle and increased SFE of the PET surface with the lipid monolayers due to specific organisation of molecules within the chitosan scaffold. The most promising combination of surface modification for tissue engineering applications seems to be the PETair/Ch/DPPC-Chol system.