Undecaprenyl pyrophosphate synthase (UPPS) is an enzyme essential for bacterial viability. It catalyzes the sequential condensations of eight isopentenyl pyrophosphate molecules with farnesyl pyrophosphate to form C55 undecaprenyl pyrophosphate, which is the lipid carrier for the precursors of various cell wall structures, such as peptidoglycan, teichoic acids, and O-antigens. The critical biological function makes UPPS an attractive target for the discovery of novel antibacterial agents. In a high throughput screening, hits with a tetramic acid core structure were identified as specific inhibitors of UPPS. These compounds possess activities against key Gram-positive pathogens including Staphylococcus aureus, Streptococcus pneumonia, and Enterococcus faecalis. Subsequent medicinal chemistry effort generated analogs with potent enzyme and whole-cell inhibitory activities. The compounds with improved activity and solubility were successfully co-crystallized with UPPS. The resulting structures map the UPPS inhibitors to an allosteric site next to the substrate binding pocket. The unique binding mode revealed by the cocrystal structures provided insight for further lead optimization.