In this study, we adopt a combination of in silico and in vitro methods to discover, validate and optimize inhibitors of PLY. We report molecules able to block hemolysis exerted by PLY and suppress cytotoxicity of PLY toward human lung cells. Using cryo-TEM tomography we show that the inhibitors efficiently inhibit pore-formation by this bacterial toxin. Mode of action, binding site, kinetics, and specificity of inhibitors are validated by applying biolayer interferometry and protein mass spectrometry with wild-type and mutant PLY as well as with other proteins. Finally, the physiological efficacy of the best inhibitor is demonstrated by using an in vitro bacterial infection model in human lung epithelial cells.