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Plant parasitic nematodes (PPNs) are highly destructive and difficult to control, while conventional chemical nematicides are highly toxic and cause serious environmental pollution. Additionally, resistance to existing pesticides is becoming increasingly common. Biological control is the most promising method for the controlling of PPNs. Therefore, the screening of nematicidal microbial resources and the identification of natural products are of great significance and urgency for the environmentally friendly control of PPNs. In this study, the DT10 strain was isolated from wild moss samples and identified as <i>Streptomyces</i> sp. by morphological and molecular analysis. Using <i>Caenorhabditis elegans</i> as a model, the extract of DT10 was screened for nematicidal activity, which elicited 100% lethality. The active compound was isolated from the extracts of strain DT10 using silica gel column chromatography and semipreparative high-performance liquid chromatography (HPLC). The compound was identified as spectinabilin (chemical formula C₂₈H₃₁O₆N) using liquid chromatography mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Spectinabilin exhibited a good nematicidal activity on <i>C. elegans</i> L1 worms, with a half-maximal inhibitory concentration (IC₅₀) of 2.948 μg/mL at 24 h. The locomotive ability of <i>C. elegans</i> L4 worms was significantly reduced when treated with 40 μg/mL spectinabilin. Further analysis of spectinabilin against known nematicidal drug target genes in <i>C. elegans</i> showed that it acts via target(s) different from those of some currently used nematicidal drugs such as avermectin and phosphine thiazole. This is the first report on the nematicidal activity of spectinabilin on <i>C. elegans</i> and the southern root-knot nematode <i>Meloidogyne incognita</i>. These findings may pave the way for further research and application of spectinabilin as a potential biological nematicide.