Bacterial species of the genus Lysobacter are environmentally ubiquitous with strong antifungal biocontrol potential.  Heat-stable antifungal factor (HSAF) secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 has broad-spectrum and highly efficient antifungal activity.  Studying the biosynthetic regulations of HSAF would lay an important foundation for strain engineering toward improved HSAF production.  In this work, we demonstrate that Le0752, an orotidine-5´-phosphate decarboxylase enzyme (ODCase) catalyzing a pivotal step of the UMP de novo biosynthesis pathway, is vital for HSAF-mediated antimicrobial activities and growth of L. enzymogenes OH11, but not for twitching motility.  This gene regulates the production of HSAF by affecting the expression of lafB, a key gene in the HSAF biosynthesis operon, through the transcription factor Clp.  Interestingly, bioinformatics analysis revealed that Le0752 belongs to the Group III ODCases, whereas its homologs in the closely related genera Xanthomonas and Stenotrophomonas belong to Group I, which contains most ODCases from Gram-positive bacteria, Gram-negative bacteria and cyanobacteria.  Moreover, the Group I ODCase PXO_3614 from the Xanthomonas oryzae pv.  oryzae PXO99^A strain complemented the Le0752 mutant in regulating HSAF-mediated antagonistic activity.  Together, these results highlight the important requirement of de novo pyrimidine biosynthetic enzymes for antibiotic HSAF production in L. enzymogenes, which lays an important foundation for improving HSAF production via metabolic flow design and for dissecting the regulatory functions of bacterial ODCases.