(Chemical Equation Presented) (E)-5-Nitro-6-(2-hydroxystyryl)pyrimidine-2, 4(1H,3H)-dione (9) was identified as a novel inhibitor of Schizosaccharomyces pombe lumazine synthase by high-throughput screening of a 100000 compound library. The Ki of 9 vs Mycobacterium tuberculosis lumazine synthase was 95 μM. Compound 9 is a structural analogue of the lumazine synthase substrate 5-amino-6-(D-ribitylamino)-2,4-(1H,3H)pyrimidinedione (1). This indicates that the ribitylamino side chain of the substrate is not essential for binding to the enzyme. Optimization of the enzyme inhibitory activity through systematic structure modification of the lead compound 9 led to (E)-5-nitro-6-(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (26), which has a Ki of 3.7 μM vs M. tuberculosis lumazine synthase.