Arthropod-borne flavivirus infection causes serious morbidity and mortality worldwide, but there are currently no effective antiflaviviral chemotherapeutics available for human use. Therefore, it is critical that new therapeutics against virus-specific targets be developed. To identify new compounds that may be used as broadly active flavivirus therapeutics, we have performed a high-throughput screening of 235,456 commercially available compounds for small-molecule inhibitors of the dengue virus NS5 RNA capping enzyme. We identified a family of compounds, the 2-thioxothiazolidin-4-ones, that show potent biochemical inhibition of capping enzyme GTP binding and guanylyltransferase function. During the course of structure-activity relationship analysis, a molecule within this family, (E)-{3-[5-(4-tert-butylbenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]propanoic acid} (BG-323), was found to possess significant antiviral activity in a dengue virus subgenomic replicon assay. Further testing of BG-323 demonstrated that this molecule is able to reduce the replication of infectious West Nile virus and yellow fever virus in cell culture with low toxicity. The results of this study describe the first inhibitor that targets the GTP-binding/guanylyltransferase activity of the flavivirus RNA capping enzyme.