Abstract Background Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. This process can be applied in bioelectrochemical systems in which the organisms produce an electrical current by respiring with an anode as electron acceptor. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalyzed by the terminal reductases and in some cases as endogenous electron shuttles. Results In the model organism Shewanella oneidensis , riboflavin production and excretion trigger a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum-sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescine decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations, which by far exceed the necessary threshold value. Conclusion This study describes a new quorum-sensing mechanism in S. oneidensis . Biofilm formation of S. oneidensis is induced by low concentrations of riboflavin resulting in an upregulation of the ornithine-decarboxylase speC . The results can be applied for the development of strains catalyzing increased current densities in bioelectrochemical systems.