Dethiobiotin synthetase (DTBS), the penultimate enzyme in biotin biosynthesis, is proposed as a promising new target for anti-TB drug development due to its essential role in tuberculi virulence and pathogenesis. The X-ray crystal structure of the enzyme from Mycobacterium tuberculosis (MtDTBS) in complex with CTP was employed for virtual screening of two compound libraries, 93,904 fragments and 57 CTP analogues. Compounds were ranked based on their predicted binding affinity and the top-ranked commercially available hits were purchased for further analysis. Surface plasmon resonance (SPR) and DTBS in vitro enzyme assays were performed in parallel to assess the potency of binding and inhibition, respectively. A total of six hits, including the known anti-cancer drug gemcitabine, showed encouraging ligand efficiency (0.2 - 0.3 kcal/mol/heavy atom). In silico docking studies predicted binding of hits in the phosphate-binding loop between the binding pockets for DAPA and NTP. This was confirming by Lineweaver-Burk analysis that showed competitive binding of gemcitabine against both DAPA and MgATP. The identified “hot-spot” for inhibitor binding, and the variety of chemical structures predicted here, provide valuable starting points for the optimization of new lead compounds for anti-TB development.