Lysobactin, also known as katanosin B, is a potent antibiotic with in vivo efficacy against Staphylococcus aureus and Streptococcus pneumoniae. It was previously shown to inhibit peptidoglycan (PG) biosynthesis, but its molecular mechanism of action has not been established. Using enzyme inhibition assays, we show that lysobactin forms 1:1 complexes with Lipid I, Lipid II, and Lipid II(A)(WTA), substrates in the PG and wall teichoic acid (WTA) biosynthetic pathways. Therefore, lysobactin, like ramoplanin and teixobactin, recognizes the reducing end of lipid-linked cell wall precursors. We show that despite its ability to bind precursors from different pathways, lysobactin's cellular mechanism of killing is due exclusively to Lipid II binding, which causes septal defects and catastrophic cell envelope damage.