Structure-based virtual screening provided a short list of 10 virtual hits from a library of 555,000 compounds for in vitro testing. Thereof, PB-1 was identified as the most active inhibitor by using a hemolysis assay with an IC50 of 38 µM. Structural and functional optimization of PB-1 led to the development of PB-2 with 8-fold increased inhibitory potency. PB-3 was obtained through a systematic variation with further improved potency of 3 µM, and with higher chemical stability and solubility. This permitted application in cell-based assays and PB-3 was established as the first small-molecule inhibitor which was able to protect human lung alveolar cells against PLY. Inhibition of membrane insertion of wild-type PLY and pore formation were demonstrated using cryo-transmission electron microscopy and tomography. While PLY added to cholesterol-containing liposomes led to the formation of PLY-pores which could be visualized in the lipid bilayer and isolated through solvation with suitable detergents, addition of inhibitor prevented pore formation and triggered significant protein precipitation.