Rhytidenone A (1), a unique spirobisnaphthalene with a 1,7-dioxaspiro[4,4]nonan-2-one motif, and five new spirobisnaphthalenes, rhytidenones B-F (2-6), were isolated from the extract of a cultured fungal endophyte, Rhytidhysteron sp. AS21B. Their structures were elucidated mainly by analysis of NMR spectroscopic data. The structure and configuration of 1 were further confirmed by single-crystal X-ray diffraction analysis. Compounds 3 and 4 exhibited significant inhibitory activity against nitric oxide production from activated macrophages with IC50 values of 0.31 and 3.60 μM, respectively.