Three thiodiketopiperazines, botryosulfuranols A-C (1–3) were isolated from the endophytic fungus Botryosphaeria mamane. The three compounds present sulfur atoms on α- and β-positions of phenylalanine derived residues and unprecedented two spirocyclic centers at C-4 and C-2′. Their planar structures were determined by spectroscopic analysis and absolute configurations were achieved by X-ray diffraction analysis and ECD and NMR chemical shifts calculations. Botryosulfuranol A (1) was the most cytotoxic compound against four cancer cell lines (HT-29, HepG2, Caco-2, HeLa) and two healthy cell lines (IEC6, Vero) highlighting the importance of an electrophilic center for cell growth inhibition.