Abstract Three new 3,6‐dioxygenated diketopiperazines, lepistamides A–C ( 1 – 3 ), along with a known compound, diatretol ( 4 ), were isolated from the mycelial solid cultures of the basidiomycete Lepista sordida. Their structures were elucidated by spectroscopic means. The isolated diketopiperazines were evaluated for the cytotoxic activity against Astc‐a‐1, Bel 7402, and HeLa cell lines, and their biogenetic pathway was discussed.