Ozone exposure from environmental smog has been implicated as a risk factor for developing dry eye disease (DED). The tear film lipid layer (TFLL), which is the outermost layer of the tear film and responsible for surface tension reduction while blinking, is in direct contact with the environment and serves as the first line of defense against external aggressors such as environmental pollution. The impact of exposure to ozone on the biophysical properties of three TFLL model membranes was investigated. These model membranes include a binary mixture of cholesteryl oleate (CO) and L-α-phosphatidylcholine (egg PC), a ternary mixture of CO, glyceryl trioleate (GT) and PC, as well as a quaternary mixture of CO, GT, a mixture of free fatty acids palmitic acid and stearic acid (FFAs) and PC. Biophysical impacts were evaluated as changes to the surface activity, respreadability, morphology and viscoelastic properties of the films. Expansion to higher molecular areas was observed in all the TFLL model membrane films which is attributable to the accommodation of the cleaved chains in the film. Significant morphological changes were observed, namely fluidization and the disruption of the phase transition behaviour of GT, and multilayer formation of CO. This fluidization reduces the hysteresis loops for the model membranes. On the other hand, the viscoelastic properties of the films exhibited differential impacts from ozone exposure as a function of composition. These findings are correlated to chemical changes to the lipids determined using ESI-MS.