Bacterial lifestyle in multicellular aggregates is a widespread phenomenon that provides protection against host innate immune factors, antibiotics and other antibacterial agents. The antigen 43 (Ag43) autotransporter (AT) is one of the key surface determinants of aggregation in Escherichia coli and a model protein for molecular studies of aggregative behaviour. Ag43 is composed of a surface exposed passenger domain anchored to the outer membrane via its β-barrel translocator domain. Ag43 is found in all E. coli pathotypes, including uropathogenic E. coli (UPEC). UPEC is the major etiological agent of urinary tract infection (UTI) and increasingly associated with antibiotic resistance, leading to high rates of UTI treatment failure and the need to develop alternative treatment and prevention strategies. In UPEC, Ag43 mediates aggregation, biofilm formation and long-term colonization of the mouse urinary tract. Our recent structural analysis of the functional passenger domain of Ag43 revealed that its unique L-shaped structure drives the formation of cell aggregates via a molecular Velcro-like mechanism. In this study, we identified a monoclonal antibody, 10C12, that inhibits Ag43-mediated cell aggregation. The recombinant fragment antigen-binding (Fab) domain of 10C12 exhibited similar cell aggregation inhibition capacity as the 10C12 monoclonal antibody. The 10C12 Fab also inhibited Ag43-mediated UPEC biofilm formation. The 10C12 Fab binds to Ag43 with an equilibrium dissociation constant of 20 nM. Epitope mapping of the 10C12 Fab revealed that the exposed beta hairpins at the L-shaped bend of Ag43, required for the maintenance of a cell aggregation competent state, are the target of the 10C12 Fab. Co-crystalization of studies determined the precise molecular interactions between the 10C12 Fab and Ag43, and thus revealed its mechanism of inhibition. This work represents a powerful and promising approach toward the development of a novel agent for the prevention of UPEC aggregation and biofilm formation.