Prevention of catheter-associated urinary tract infection (CAUTI) over long-term usage of urinary catheters remains a great challenge. Bacterial interference using nonpathogenic bacteria, such as E. coli 83972, have been investigated in many pilot-scale clinical studies as a potentially nonantibiotic based strategy for CAUTI prevention. We have demonstrated that preforming a dense and stable biofilm of the nonpathogenic E. coli greatly enhances their capability to prevent pathogen colonization. Such nonpathogenic biofilms were formed by E. coli 83972 expressing type 1 fimbriae (fim+ E. coli 83972) on mannoside-presenting surfaces. In this work, we report the synthesis of a series of mannoside derivatives with a wide range of binding affinities, all being equipped with a handle for covalent attachment to silicone surfaces. We established a high-throughput competitive assay based on mannoside-modified particles and flow-cytometry to directly measure the binding affinity between the mannoside ligands and fim+ E. coli 83972. We demonstrated that the bacterial adhesion and biofilm formation were strongly correlated to the binding affinity of the immobilized mannoside ligands. Mass spectrometry based proteomic analysis indicated a substantial difference in the proteome of the extracellular polymeric substance (EPS) secreted by biofilms on different mannoside surfaces, which might be related to the biofilm stability.