Inhibition of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) potentially represents a new treatment option for malaria, since P. falciparum relies entirely on de novo pyrimidine biosynthetic pathway for survival. Herein, we reported a series of pyrimidone derivatives as novel inhibitors of PfDHODH. The most potent compound 26 showed high inhibition activity against PfDHODH (IC₅₀ = 23 nM), with >400-fold species-selectivity over hDHODH. This brand new inhibitor scaffold targeting PfDHODH reported in this work might lead to the discovery of new antimalarial agents.Inhibition of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) potentially represents a new treatment option for malaria, since P. falciparum relies entirely on de novo pyrimidine biosynthetic pathway for survival. In this work, a series of brand new scaffold PfDHODH-specific inhibitors pyrimidone derivatives were obtained from docking analyses and structural optimizations. The most potent inhibitor 26 showed excellent inhibitory activity against PfDHODH (IC50 = 23 nM) and high species selectivity over hDHODH. Through SAR studies, three preferential structural fragments were essential for these novel potent PfDHODH inhibitors: (1) bicyclic systems such as “naphthyl-like” substituents as the hydrophobic group; (2) two carbonyl group in the dihydrofuranone ring oriented in the same direction and formed hydrogen bonds with the polar residue (Arg265); (3) a hydrogen bond donor (NH2 in compound 20 and 26) was important for the inhibitory activity to interact with the imidazole group of His185. The results might be valuable for the novel scaffold PfDHODH inhibitors to be developed into new antimalarial agents.