Abstract: To discover novel milbemycin-type compounds with insecticidal activity, two isomers (1 and 2) of milbemycin D with nematicidal activity were isolated and purified from the secondary metabolites of Streptomyces bingchenggensis HU176. Purification was achieved using gel column chromatography and high-performance liquid chromatography (HPLC). Based on comprehensive spectroscopic analyses (¹H NMR, ¹³C NMR, HRESI-MS) and comparison with literature data, their structures were identified as 12-desmethyl-12-ethyl milbemycin A₄ and 24-desmethyl-24-ethyl milbemycin A₄, respectively. Bioassay results showed that compounds 1 and 2 exhibited moderate nematicidal activity against Bursaphelenchus xylophilus, with LC₅₀ values of 14.53 and 16.89 μg/mL, respectively. These values were comparable to that of the positive control avermectin B1a (LC₅₀ = 12.42 μg/mL). Notably, compound 1 is a new milbemycin analog, and compound 2, a known compound, is reported as a natural product for the first time. These findings provide a valuable reference for the further development and utilization of milbemycin-type compounds.