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Abstract: Investigation of the entomogenous fungus Setosphaeria rostrateLGWB-10 from Harmonia axyridisled to the isolation of four new isocoumarin derivatives, setosphlides A–D (1–4), and four known analogues (5–8). Their planar structures and the relative configurations were elucidated by comprehensive spectroscopic methods. The absolute configurations of isocoumarin nucleus for 1–4were elucidated by their ECD spectra. The C-10 relative configurations for the pair of C-10 epimers (1and 2) were established by comparing the magnitude of the computed 13C NMR chemical shifts (Δδcalcd.) with the experimental 13C NMR values (Δδexp.) for the epimers. All of the isolated compounds (1–8) were evaluated for their cytotoxicities against four human tumor cell lines MCF-7, MGC-803, HeLa, and Huh-7. Graphic Abstract: