Prenylemestrins A and B (1and 2, respectively), two unusual epipolythiodioxopiperazines featuring a thioethanothio bridge instead of a polysulfide bridge, were isolated from the fungus Emericellasp. CPCC 400858 guided by genomic analysis. Their structures were determined by extensive spectroscopic data, NMR and ECD calculations, and X-ray diffraction analysis. A plausible biosynthetic pathway for 1and 2was proposed on the basis of gene cluster analysis. Prenylemestrins A and B exhibited cytotoxicities against human chronic myelocytic leukemia cell lines K562 and MEG-01.