Abstract 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. Graphical abstract Three undescribed thiodiketopiperazines exhibiting two spirocyclic centers were extracted from the culture of the endophytic fungus Botryosphaeria mamane isolated from Bixa orellana leaves. Image 1 Highlights • Three thiodiketopiperazines were isolated from Botryosphaeria mamane. • These alkaloids possess unprecedent spiro[isoxazolidino-cyclohexenone] scaffold. • Botryosulfuranol A inhibited the growth of both cancer and healthy cell lines. [ABSTRACT FROM AUTHOR]