Polyhexamethylene guanidine phosphate (PHMG-p) is an active ingredient of humidifier disinfectants and causes severe lung injury resulting in pulmonary fibrosis. Current evidence indicates that pulmonary fibrosis is initiated as a result of epithelial damage, which can lead to an inflammatory response and fibrotic cell infiltration; however, the toxic mechanism of PHMG-p on the epithelium is still unknown. In this study, the toxic response of PHMG-p on human lung epithelial cells was evaluated, and its mechanisms associated with reactive oxygen species (ROS), DNA damage, and its relationship with p53 activation were investigated. The toxic responses of epithelial cells were assessed by flow cytometry analysis and western blot analysis. The results revealed that PHMG-p induced G1/S arrest and apoptosis in A549 cells. Interestingly, p53 was activated by PHMG-p treatment and p53 knockdown suppressed PHMG-p-induced apoptosis and cell cycle arrest. PHMG-p promoted ROS generation and consequently increased the expression of DNA damage markers such as ATM and H2AX phosphorylation. The antioxidant N-acetylcysteine reduced the expression of phosphorylated ATM and H2AX, and the ATM inhibitor, caffeine, inhibited p53 activation. Taken together, our results demonstrate that PHMG-p triggered G1/S arrest and apoptosis through the ROS/ATM/p53 pathway in lung epithelial cells.