Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosis (TB), an ancient disease which causes 1.5 million deaths worldwide. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the MTB de novo pyrimidine biosynthesis pathway, and it is essential for MTB growth in vitro, hence representing a promising drug target. We present: (i) the biochemical characterization of the full‐length MTB DHODH, including the analysis of the kinetic parameters, and (ii) the previously unreleased crystal structure of the protein that allowed us to rationally screen our in‐house chemical library and identify the first selective inhibitor of mycobacterial DHODH. The inhibitor has fluorescence properties, potentially instrumental to in cellulo imaging studies, and exhibits an IC50 value of 43 μm, paving the way to hit‐to‐lead process. Dihydroorotate dehydrogenase (DHODH) is a key enzyme of the pyrimidine biosynthesis pathway in Mycobacterium tuberculosis. Annotated as an essential gene for mycobacterial growth, it represents a potential drug target. Here, we present the crystal structure of the full‐length DHODH along with its biochemical characterization. Structural analysis has driven the screening for the identification of the first inhibitor of mycobacterial DHODH.