Human dihydroorotate dehydrogenase (<i>h</i>DHODH) is an attractive tumor target essential to de novo pyrimidine biosynthesis. Novel potent <i>h</i>DHODH inhibitors with low toxicity are urgently needed. Herein, we demonstrate the isolation of 25 ascochlorin (ASC) derivatives, including 13 new ones, from the coral-derived fungus <i>Acremonium sclerotigenum</i>, and several of them showed pronounced inhibitions against <i>h</i>DHODH and triple-negative breast cancer (TNBC) cell lines, MDA-MB-231/-468. Interestingly, we found that <i>h</i>DHODH is required for proliferation and survival of TNBC cells, and several ASCs significantly inhibited TNBC cell growth and induced their apoptosis via <i>h</i>DHODH inhibition. Furthermore, the novel and potent <i>h</i>DHODH inhibitors (<b>1</b> and <b>21</b>) efficiently suppressed tumor growth in patient-derived TNBC xenograft models without obvious body weight loss or overt toxicity in mice. Collectively, our findings offered a novel lead scaffold as the <i>h</i>DHODH inhibitor for further development of potent anticancer agents and a potential therapeutic strategy for TNBC.