Our continued bioassay-directed fractionation and purification of the mother liquor from Streptomyces griseus var. autotrophicus yielded griseulin, a novel aromatic nitro-containing compound with nematocidal and mosquitocidal activities. Structural analysis via UV, ¹H/¹³C NMR, and LRMS revealed griseulin contains a 1,2-pyrone moiety (similar to luteoreticulin), three methyl groups, one methoxy group, no furan ring, and a molecular ion at m/z 341 (C₁₉H₁₉O₅N), consistent with its proposed structure. Under identical fermentation conditions, Streptomyces spectabilis (ATCC 27465), Streptomyces luteoreticuli, and Streptoverticillium mobaraense (ATCC 25365) also produced griseulin. Bioassays demonstrated griseulin caused 100% mortality in Panagrellus redivivus, Caenorhabditis elegans, and Heterodera glycines at 5 ppm (4 hours) and 1 ppm (24 hours), respectively, and 100% mortality in 4th instar Aedes aegypti larvae within 24 hours. Addressing the limitations of expensive, environmentally toxic organophosphate nematicides (for which no eradicative soil nematode treatments exist), griseulin represents a promising candidate for developing safer, effective nematocidal agents.