Plasmodium knowlesi has been recently recognized as a human malarial parasite, particularly in the region of south-east Asia. The effective prevention and treatment of this disease is increasingly bound to fail due to the emergence of drug resistance. Hence, design of new drugs against known targets is gaining importance. Pyrimidine pathway is a crucial metabolic pathway in P. knowlesi, and the enzymes involved are also unique in terms of their structure and function as compared to its human counterpart. Thus targeting Dihydroorotase, an enzyme involved in the pyrimidine pathway, provides a promising route for novel drug development. The 3D structure of P. knowlesi Dihydroorotase is not available. The structural homologues of the enzyme are not available in the database, so a threading approach is used to predict the structure. The steric clashes of the predicted model are removed by running a MD simulation of 20 ns. Then the resulting structure is validated by using Ramachandran plot and G-factor analysis. The active sites are predicted and they show interactions with His13, His15, Asp256, Lys97, His134 and His169 for two Zn atoms, and Arg17, Asn42, Thr43, Pro100, His260 and Lys271 for the Dihydroorotate. Interactions between the ligand and binding pocket residues are extracted to create a structure-based pharmacophore model of the docked complex. A four point based pharmacophore model, with four H-bond acceptors and one negative carboxyl ion, was used as a 3D query for screening against 2,664,779 standard lead compounds, obtained from freely available ZINC database. Top 15 compounds with higher pharmacophore-fit score were considered for further study. Among these, only four compounds show desired drug-like properties, and follow the Lipinski's rule of five. Two compounds (ZINC22066495, ZINC20136046) that are negatively charged are found to be more suitable for interaction with positively charged active site of enzyme. Molecular dynamics simulation is used to check the stability of negatively charged two compounds along with natural compound at the active site of DHOase. Results of simulation were analyzed in terms of RMSD, Rg and RMSF analysis. The ZINC22066495-DHOase complex shows stable interaction throughout the simulation compared with the other two complexes. This lead compound (ZINC22066495) with novel scaffolds, may act as a potential drug for treating malaria caused by P. knowlesi.