A prefractionated Streptomyces-derived extract was initially identified as being active using a luciferase-based AMP-activated protein kinase (AMPK) assay. Bioassay-guided fractionation led to the isolation of the new compound quinazolin-4(3H)-one (1) as the active component. However, 1 was shown to have potent firefly luciferase inhibitory activity with no effect on AMPK. This is the first report of a natural luciferase inhibitor.