In the present study, stability and structure of single and binary mixed monolayers of an unsaturated phospholipid, DOPC, and a saturated phospholipid, DPPC-d75, on the water surface, were explored using the π-A isotherm, atomic force microscopy (AFM) and sum frequency generation (SFG) vibrational spectroscopy in various environments. Our results demonstrated that DOPC in the monolayers becomes unstable after the exposure to a low concentration of ozone (20 ± 10 ppb) or even to ambient laboratory air, which has a similar ozone level, but is stable in nitrogen or oxygen. DOPC can be selectively oxidized by a trace amount of ozone in the ambient environment but can be partially inhibited by the presence of DPPC in the monolayer. The present study provides useful information for understanding the physicochemical properties of the cell membranes.