Squalene (SQ) possesses a wide range of pharmacological activities (antioxidant, drug carrier, detoxifier, hydrating, emollient) that can be of benefit to the ocular surface. It can come in contact with human meibum (hMGS; the most abundant component of the tear film lipid layer) as an endogenous tear lipid or from exogenous sources as eyelid sebum or pharmaceuticals. The aims of this study were to determine (i) if SQ is in tear lipids and (ii) its influence on the surface properties of hMGS films. Heteronuclear single quantum correlation NMR confirmed 7 mol % SQ in Schirmer’s strips extracts. The properties of SQ/hMGS pseudo-binary films at the air/water interface were studied with Langmuir surface balance, stress-relaxation dilatational rheology and Brewster angle microscopy. SQ does not possess surfactant properties. When mixed with hMGS squalene (i) localized over the layers’ thinner regions and (ii) did not affect the film pressure at high compression. Therefore, tear SQ is unlikely to instigate dry eye, and SQ can be used as a safe and “inert” ingredient in formulations to protect against dry eye. The layering of SQ over the thinner film regions in addition to its pharmacological properties could contribute to the protection of the ocular surface.