With the intention to move away from animal testing for the toxicological evaluation of chemicals comes the need to develop new approach methodologies which are mechanism-anchored and target relevant key events leading to an adverse outcome. To date, no validated alternative methods are available for studying the acute inhalation toxicity potential of airborne chemicals but the constrained drop surfactometer measuring the surface tension of a drop of lung surfactant presents as a promising candidate. Indeed, the correlation of the increase in minimum surface tension of lung surfactant in vitro with changes in the breathing patterns of mice after inhalation of test compounds has been shown in multiple studies. However, the causal factors leading to lung surfactant inactivation remain speculative. This paper combines molecular and biophysical methods (constrained drop and captive bubble surfactometers, Langmuir-Blodgett balance, epifluorescence microscopy, cryogenic transmission electron microscopy, and differential scanning calorimetry) applied to purified porcine lung surfactant and dipalmitoylphosphatidylcholine interfacial films to gain insights into the disruption of lung surfactant function by three chemicals known to show acute inhalation toxicity (trimethoxyoctylsilane, methyl 3-oxo-2-pentylcyclopentaneacetate, and diisopentyl ether). The results of this study suggest that the test chemicals intercalate between the phospholipids at the air-liquid interface, reduce the stability of the films, and decrease the cohesivity of interface-associated multilayered structures thereby perturbing the lung surfactant surface activity. These findings contribute to a better understanding of chemically-induced lung surfactant function disruption.