Bacterial biofilm formation is thought to enhance survival in natural environments and during interaction with hosts. A robust colonizer of the human gastrointestinal tract, Escherichia coli Nissle 1917, is widely employed in probiotic therapy. In this study, we performed a genetic screen to identify genes that are involved in Nissle biofilm formation. We found that F1C fimbriae are required for biofilm formation on an inert surface. In addition, these structures are also important for adherence to epithelial cells and persistence in infant mouse colonization. The data suggest a possible connection between Nissle biofilm formation and the survival of this commensal within the host. Further study of the requirements for robust biofilm formation may improve the therapeutic efficacy of Nissle 1917.