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Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of d-leucine, d-methionine, d-tyrosine, and d-tryptophan that could act at nanomolar concentrations. d-Amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of d-Amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. d-Amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. d-amino acids are produced by many bacteria and, thus, may be a widespread signal for biofilm disassembly.