Polymicrobial biofilms are ubiquitous, and the complex interspecies interactions within them are cryptic. We discovered the chemical foundation of antagonistic interactions in a model dual-species biofilm in which Pseudomonas aeruginosa inhibits the biofilm formation of Agrobacterium tumefaciens. Three known siderophores produced by P. aeruginosa (pyoverdine, pyochelin, and dihydroaeruginoic acid) were each capable of inhibiting biofilm formation. Surprisingly, a mutant that was incapable of producing these siderophores still secreted an antibiofilm metabolite. We discovered that this inhibitor was N5-formyl-N5-hydroxy-l-ornithine (fOHOrn)─a precursor in pyoverdine biosynthesis. Unlike the siderophores, this inhibitor did not appear to function via extracellular metal sequestration. In addition to this discovery, the compensatory overproduction of a new biofilm inhibitor illustrates the risk of pleiotropy in genetic knockout experiments. In total, this work lends new insight into the chemical nature of dual-species biofilm regulation and reveals a new naturally produced inhibitor of A. tumefaciens biofilm formation.