Lysobacter enzymogenes OH11 is currently considered to be a novel biocontrol agent for various plant fungi diseases. At present, only heat-stable antifungal factor (HSAF) has been isolated and identified in culture, although other active compounds also showed antifungal activity. In the present study, a novel active compound, alteramide B (ATB), which exhibits broad-spectrum antagonistic activity against phytopathogenic fungi and oomycetes, was isolated. The genes responsible for ATB biosynthesis were also determined. In addition, a strain producing ATB with minimal HSAF production was successfully generated by redirecting metabolic flux, namely L. enzymogenes OH57. Furthermore, ATB production increased to 893.32 15.57 mg/L through medium optimization and precursor supply strategy, which was 24.36-fold higher than that of 10% tryptic soy broth (36.67 1.63 mg/L). Taken together, this study indicates ATB has great development value as a biopesticide because of its bioactivity and high production.