Five novel perhydrobenzannulated 5,5-spiroketal sesquiterpenes, namely, pleurospiroketals A-E (1-5), were isolated from the culture of the edible mushroom Pleurotus cornucopiae. Pleurospiroketals D (4) and E (5) were obtained as an isomeric mixture with a ratio of 5:4. Their structures were established by NMR, X-ray single-crystal diffraction, and CD data analysis. Pleurospiroketals A-E (1-5) are sesquiterpenoids with a unique benzannulated 5,5-spiroketal skeleton. Compounds 1-3 showed inhibitory activity against nitric oxide production in lipopolysaccharide-activated macrophages with IC(50) values of 6.8, 12.6, and 20.8 μM, respectively.