Langmuir films of amphiphilic biomolecules represent promising systems to study diffusional properties in model membranes under controlled area conditions. Here, we present for the first time single molecule fluorescence imaging experiments on phospholipid monolayers at the air-water interface. The technique is used to track the lateral diffusion of single molecules of N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoe thanolamine, triethylammonium salt (TRITC-DHPE), in phospholipid monolayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DMPG) at different areas per phospholipid molecule. Our tracking data of the averaged mean-square displacement indicate for both phospholipids that surface flow could be suppressed significantly. The diffusion behavior of TRITC-DHPE in DMPC and DMPG monolayers is characterized by unobstructed diffusion and can be described well by a free area model. Our experiments show that single molecule fluorescence imaging can be successfully applied to monolayers of amphiphiles at the air-water interface. This opens the door to future studies of hindered diffusion in monolayers of specific heterogeneity.