A high throughput screen was conducted to identify inhibitors of the RNA-dependent RNA polymerase (RdRp) of HCV. One of the classes identified was related to toxoflavin, a naturally occurring antibiotic. These compounds were potent inhibitors of HCV RdRp, with optimization of the initial hits giving compounds with half-maximal inhibition at 200 nM. However, binding studies indicated that a physical interaction with RdRp was too weak to be detected. Inhibition was dependent on reducing agent, implicating the flavin core as essential to the inhibitory properties of these compounds. The accumulation of the inhibitory species occurred over a time span of 1 to 2 hours, the period of time required for oxidation of a 10 to 100-fold excess of DTT by the flavin. These results indicate that the inhibitory species is likely to be a chemically reactive species resulting from the interaction of the flavin and DTT. Human RNA polymerase II was also sensitive to inhibition by these toxoflavin-like compounds. While this class of inhibitors has generated interest as antibacterial, antifungal and antitumor agents, their use has been limited by toxicity. These results provide a potential mechanism to explain the toxicity of these compounds.