Moenomycin inhibits bacterial growth by clocking the transglycosylase activity of class A penicillin-binding proteins (PBPs), which are key enzymes in bacterial cell wall synthesis. The binding affinities of moenomycin A with various truncated PBPs were compared showing that the transmembrane domain is important for moenomycin binding. Full-length class-A PBPs from 16 bacterial species were produced, and their binding activities showed a correlation with the antimicrobial activity of moenomycin against Enterococcus faecalis and Staphylococcus aureus. Moreover, a fluorescence anisotropy-based high-throughput assay was developed and used successfully for identification of transglycosylase inhibitors.