Fire blight, caused by Erwinia amylovora, devastates rosaceous plants. Erwinia herbicola (syn. Pantoea agglomerans) colonizes the same plant surfaces and produces antibiotics to control E. amylovora, but its antibiotics are complex (multiple components in single strains, different strains produce different complexes, activity suppressed by amino acids), hindering characterization. As a first step, we used cloning and heterologous expression of individual biosynthetic pathways to decipher one antibiotic's molecular structure and genetics. A cosmid library of E. herbicola strain 318 (Eh318) was constructed in Escherichia coli, identifying two antibiotic-producing clones: pantocin A (activity suppressed by histidine) and B (suppressed by arginine). Here, we report pantocin B's isolation, structure determination, and mechanism of action. Pantocin B was isolated from large-scale fermentation of subclone pCPP719 (1 mg/L culture) via cation- and anion-exchange chromatography and HPLC. In minimal media, it has picomolar activity against E. amylovora (IC50 = 750 pM). Its molecular formula (C9H17N3O6S) was established by high-resolution FAB MS. Planar structure was determined via 1D/2D 1H and 13C NMR, with relative/absolute stereochemistry confirmed by single-crystal X-ray diffraction and chiral HPLC of L-alanine hydrolysis product. Pantocin B inhibits E. amylovora N-acetylornithine transaminase, competitive with N-acetylornithine (Ki ≈ 250 µM) and uncompetitive with glutamine. Biosynthetically, its N-terminus derives from alanine, and the methylenediamine may come from glycine. Mutant studies showed Eh318 mutants deficient in pantocin B (Eh439) or A (Eh421) alone still control E. amylovora, but the double mutant (Eh440) is markedly less effective. This study demonstrates the power of cloning/heterologous expression for isolating unknown natural products, and pantocin A/B are major contributors to Eh318's biocontrol ability.