The discovery of new inhibitors of juvenile hormone (JH) biosynthesis has attracted much attention in the last 20 years because of their potential use as commercial insecticides, especially inhibition of the final steps of JH biosynthesis (methyl farnesoate formation and epoxidation) since the involved enzymes are specific for insect isoprenoid biosynthesis. We report here the isolation and identification of brevioxime from Penicillium brevicompactum, a new compound with an unusual heterocyclic oxime structure that exhibits very high activity as a JH biosynthesis inhibitor. A systematic screening of 118 Penicillium strains from cereal contamination led to selecting a P. brevicompactum strain; its dichloromethane extract showed high entomotoxicity to Oncopeltus fasciatus and blocked spontaneous JH biosynthesis in vitro. Bioassay-guided fractionation via silica gel column chromatography and HPLC yielded pure brevioxime, whose structure was determined by spectral data and X-ray diffraction. Brevioxime completely blocked JH production in Locusta migratoria CA at 100 µM (60% inhibition at 0.5 µM), and late intermediates (farnesol, farnesoic acid) did not restore JH production, indicating inhibition of final steps (methylation/epoxidation). It also fully inhibited JH epoxyacid production in Agrotis ipsilon male CA at 100 µM, suggesting it may inhibit P450-linked enzymes. Isolation of brevioxime illustrates the potential of fungi as a source of active molecules for developing biorational insecticides.