Nucleotide-glycosyltransferases (NDP-Gtfs) play key roles in a wide range of biological processes. It is difficult to probe the roles of individual glycosyltransferases or their products because, with few exceptions, selective glycosyltransferase inhibitors do not exist. Here, we investigate a high-throughput approach to identify glycosyltransferase inhibitors based on a fluorescent donor displacement assay. We have applied the screen to E. coli MurG, an enzyme that is both a potential antibiotic target and a paradigm for a large family of glycosyltransferases. We show that the compounds identified in the donor-displacement screen of MurG are selective for MurG over other enzymes that use similar or identical substrates, including structurally related enzymes. The donor displacement assay described here should be adaptable to many other NDP-Gtfs and represents a new strategy to identify selective NDP-Gtf inhibitors.