To perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents. The interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air/water interface were examined in vitro during controlled compression/expansion of the film area by Langmuir surface balance, surface potential measurements, and pendant drop - axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the sample's lateral elasticity and capability to compress and spread during dynamic area changes. Lipid film morphology was monitored by Brewster Angle microscopy. The viability of BAC-treated Statens Seruminstitut Rabbit Cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001-0.02%. In the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in a) impaired lipid spread and formation of discontinuous non-uniform surface layers, b) increased surface pressure-area hysteresis during compression/expansion, and c) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure and their magnitude increased with BAC concentration. The surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well-explain TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.