Recently, the importance of inhalation toxicity assessment increased due to recent humidifier disinfectant-associated deaths in children. Benzalkonium chloride (BAC) is currently used as a cationic surfactant and germicide in food industry processing lines and as a hand sanitizer. Animal models are mainly used as a method of evaluating the inhalation toxicity of a hazardous substance, but that approach requires considerable amounts of time and cost. As a replacement for animal experiments, in vitro cell culture can be used to assess toxicity. However, such culture does not reflect the natural microenvironment of the lung, particularly its dynamic nature. In this study, we simulated normal breathing levels (tidal volume 10%, 0.2 Hz) through surface elongation of an elastic membrane in a dynamic culture system. The low-cost dynamic system provided easy control of breathing rate during lung cell culture. We assessed the toxicity using different concentrations of BAC (0, 2, 5, 10, 20, and 40 μg/mL) under static and dynamic culture conditions. Following 24 h of exposure to BAC, cellular metabolic activity, cell membrane integrity, interleukin-8 (IL-8) and reactive oxygen species (ROS) levels, and the total amount of protein in cells were analyzed. Our results showed that significant differences in cellular metabolic activity, as well as IL-8 and ROS profiles, between static and dynamic cell growth conditions, following BAC exposure.