Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage < 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin.