Catheter-associated urinary tract infections (CAUTIs) are frequent hospital-acquired infections, with one third of the cases caused by uropathogenic Escherichia coli (UPEC). The lectin FimH, located on type 1 fimbriae, mediates UPEC adhesion to the catheter surface initiating biofilm formation. Since blocking FimH-mediated adhesion can impede biofilm formation, we have evaluated the in vitro effectiveness of four classes of FimH inhibitors against CAUTIs. FimH antagonists successfully prevented biofilm formation in several UPEC strains, although could not eliminate established biofilms. In a novel catheter-associated biofilm model, the most potent, preventively applied antagonist (para-cyano biphenyl α-d-mannopyranoside) reduced type 1 pili-mediated biofilm mass by 50%, without affecting bacterial viability. Co-administration of FimH antagonist and ciprofloxacin resulted in an almost 3 Log₁₀ colony-forming-units (CFU) reduction of biofilm-embedded bacteria. In conclusion, when used preventively, FimH antagonists can potentiate antibiotic activity against UPEC biofilm and could serve as a valuable prophylactic measure to decrease the occurrence of CAUTIs.