The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by ramoplanin, but there is no definitive information about whether one step is inhibited preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin and the related compound enduracidin have an intrinsic preference for one step over the other. Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These results provide compelling evidence that ramoplanin's and enduracidin's primary cellular target is the transglycosylation step of peptidoglycan biosynthesis.