Two new pyranonaphthoquinones, phialoyxinones A (1) and B (2), a new eighteen-membered ring lactone, phialoyxtone (3), and five known pyranonaphthoquinone derivatives were identified from the fungus Phialocephala sp. YUD18001, which was isolated from the rhizospheric soil associated with Gastrodia elata. Their structures were unequivocally established by a comprehensive interpretation of the spectroscopic data, with the stereochemistry for 1-3 was defined by a combination of TDDFT calculations, and the DP4+ probability analysis based on NMR chemical shift calculations. All of the new compounds 1-3 were evaluated for cytotoxicity and acetylcholinesterase inhibitory, compound 2 exhibited in vitro cytotoxic activities against five human cancer cell lines (HL-60, SMMC-7721, A549, MCF-7 and SW480) with IC50 values ranging from 11.80 to 19.32 μM. Compounds 2 and 3 exhibited moderate AChE inhibitory activities. A putative biosynthetic pathway for the pyranonaphthoquinones was proposed.