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A new naphthoquinone compound named 7-hydroxyl-8-methoxyltrypethelone 3 together with two known compounds trypethelone 1 and trypethelone methyl ether 2 was isolated from cultured mycobiunt of Astrothelium sp.The structures were elucidated by 1D and 2D NMR spectroscopic analysis as well as comparison with those reported in literatures.Compound 1displayed significant antibacterial activity against Bacillus subtilis(ATCC 6633),and showed modest antibacterial activity against Staphylococcus aureus col(MRSA)(CGMCC 1.2465),while compounds 2 and 3 showed modest antibacterial activity against Enterococcus faecalis 850E(CGMCC 1.2135)and S.aureus col(MRSA)(CGMCC 1.2465).