<p>A 3:1 disinfectant composed of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT) has been widely used in water-based household products such as detergents, toothpaste, and cosmetic applications. In the United States, it was classified as a pesticide in 1998 and designated as a Category 2 dermal-toxicity substance for cosmetics. Several studies in the Republic of Korea have reported that components of humidifier disinfectants (HDs) such as polyhexamethylene guanidine (PHMG), CMIT, and MIT cause chronic pulmonary conditions. However, its toxicity is still controversial because of the discrepant results between human cases and animal experiments. We recapitulate the in vivo characteristics of the pulmonary epithelial cells and introduce an air-liquid interface (ALI) system to replicate acute inhalation injury. To assess compound-specific toxicity in the human airway, NHBE cells were first differentiated under ALI conditions and then exposed to MIT under submerged conditions. In parallel, ^AXBarrier-on-Chip (AlveoliX AG) technology integrated with the Cloud α AX12 (Vitrocell Systems GmbH) allowed us to mimic human lung physiology more realistically. In this study, this inhalation platform was utilized to nebulize MIT onto primary cell-derived immortalized alveolar epithelial cells (^AXiAECs) co-cultured with human lung microvascular endothelial cells (hLMVECs) seeded onto the AX12. Additionally, PHMG was used as a positive control to compare MIT toxicity levels. Using this approach, significant barrier disruption and cytotoxicity were observed when MIT was introduced into the alveolar lung-on-chip. Overall, we confirmed MIT-induced toxicity in both bronchial and alveolar epithelial models, highlighting the importance of multiscale in vitro systems for inhalation toxicology.</p>