AbstractA new polyaromatic metabolite, ent‐herqueidiketal (1), and a new phenalenone derivative, epi‐peniciherqueinone (2), along with twelve known compounds 3–14, were isolated from the fungus Penicillium herquei YNJ‐35, a symbiotic fungus of Pulveroboletus brunneopunctatus collected from Nangunhe Nature Reserve, Yunnan Province, China. The structures of 1–14 and the absolute configurations of 1 and 2 were determined by their spectroscopic data or by their single‐crystal X‐ray diffraction analysis or optical rotation values. Compound 1 showed strong antibacterial activity against Staphylococcus aureus (ATCC 29213) with minimum inhibitory concentration (MIC) of 8 μg/mL. In the cytotoxicity assays, compound 1 showed weak inhibitory activity against breast cancer MCF‐7 and mice microglial BV2 cells with half maximal inhibitory concentration (IC₅₀) of 17.58 and 29.56 μM; compound 14 showed stronger cytotoxicity against BV2 and MCF‐7 cells with IC₅₀ values of 6.57 and 10.26 μM.