Background: Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-bindinq FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade-bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-la and αβ1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infectin of neighbouring cells, being the major cause of the troublesome recurrent urinary tract Infections. Methodology/Principal Findings: We demonstrate that α-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl α-D-mannose prevents binding of type 1-pillated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UT189 cystitis isolate instilled in mouse bladder via catheterization. Heptyl α-D-mannose also specifically ihhibited biofilm formation at micromolar concentrations. The structural basis the great inhibitory potential of alkyl and aryl α-D-mannosides was elucidated in the cryatal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH intetacts with Manα1,3Manβ1,4GlcNAcβ1,4GlcNAc in an extended binding site. The Interactions along the α1,3 gycosidic bond and the first β1,4 linkage to the chito biose unit are conserved with those of FimH with buty α-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group. Conclusion/Significance: The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extend into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.