The novel naphthoquinone 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was created as a potential chemotherapeutic agent. Previous work showed it is an irreversible inhibitor of type I and II topoisomerases that alkylates specific enzyme thiols. While analyzing the effect of TU100 on cancer cells, we discovered it is a potent inhibitor of luciferase derived from both Photinus pyralis (fireflies) and Renilla reniformis (sea pansy). Pre-incubation experiments showed that TU100 does not irreversibly inactivate luciferase, indicating its mechanism is different from that observed with topoisomerases. Firefly luciferase generates light using ATP and luciferin as substrates (bioluminescence). An examination of TU100 inhibition at varying substrate concentrations revealed the drug is uncompetitive with respect to ATP and competitive with respect to luciferin. The TU100 binding constant (K(I)) is 2.5±0.7 μM as determined by Dixon plot analysis. These data suggest TU100 specifically binds the luciferase-ATP complex and prevents its interaction with luciferin. Given the novel structure of TU100, unique mechanism of action, and ability to target luciferase from different species, these results identify TU100 as an important new reagent for investigating and regulating bioluminescent enzymes.