Every year, cases of respiratory distress are recorded in humans following exposure to chemicals. The underlying mechanisms of toxicity remain unclear. We have previously shown that pulmonary surfactant is the target of impregnation spray products, leading to lung damage in humans. Lung surfactant is a lipid-protein mixture whose main function is to reduce the surface tension in the alveoli at the end of expiration, minimizing the work of breathing. In this study, three chemicals (trimethoxyoctylsilane, 3-oxo-2-pentyl-cyclopentaneacetic acid and diisoamyl ether), classified as acute inhalation toxicants and of relevance for worker and consumer exposure, were studied with respect to their potential to perturb lung surfactant function. Two models were used: DPPC, the most abundant surface active lipid in surfactant, as well as native surfactant purified from porcine bronchoalveolar lavage. The constrained drop surfactometer and the captive bubble surfactometer were used to assess the effects of the chemicals on the biophysical activity of the two surfactant models under different exposure conditions. These two methodologies mimic the breathing dynamics of the lungs. In addition, the effect of the chemicals on the lung surfactant structure was explored by ( i) Langmuir-Blodgett balance experiments coupled with epifluorescence microscopy, and ( ii) cryogenic transmission electron microscopy. Lastly, the thermotropic behavior of surfactant after exposure to the chemicals was investigated using differential scanning calorimetry. The data provide evidences on the mechanism of toxicity of chemicals potentially harmful for the lungs. The identification of key event(s) for the acute inhalation toxicity pathway is crucial for the design of relevant toxicity assays in vitro, which will contribute to having safer chemicals in the workplace and on the market.