A new 14-membered resorcylic acid lactone (RAL 14 ), chaetolactone A ( 1 ), along with three known ones ( 2 - 4 ), was obtained from the fermentation of the soil-derived fungus Chaetosphaeronema sp. SSJZ001. Their structures were established based on extensive spectroscopic data analyses (UV, IR, HRESIMS, 1D, and 2D NMR), 13 C NMR chemical shifts calculations coupled with the DP4 + probability method, theoretical calculations of ECD spectra, as well as X-ray diffraction analysis. All compounds were evaluated for their cytotoxic effects against A549, HO-8910, and MCF-7 cell lines.