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Three new highly oxygenated sterols were isolated from Cladosporium sp. SCSIO41007.All compounds were elucidated by spectroscopic methods.Absolute configurations were determined by X-ray single-crystal and CD methods.Compound 2 exhibited weak antiviral activity against H3N2.Three new highly oxygenated sterols (1–3) and a new dihydroisocoumarin (7) together with six known compounds were isolated from the extracts of the culture of a sponge-derived fungus Cladosporium sp. SCSIO41007. The structures of all new compounds (1–3, 7) were determined by the extensive spectroscopic analysis including NMR, MS, IR, and UV. Their absolute configurations were determined by X-ray single-crystal and CD data analysis. Compound 2 exhibited weak inhibitory activity against H3N2 with the IC50 value of 16.2μM.