Biofilm formation and host-pathogen interactions are frequently studied using multiwell plates; however, these closed systems lack shear force which is present at several sites in the host such as the intestinal and urinary tracts. Recently, microfluidic systems, which incorporate shear force and very small volumes, have been developed to provide cell biology models that resemble in vivo conditions. Therefore, the objective of this study was to determine if the BioFlux 200 microfluidic system could be used to study host-pathogen interactions and biofilm formation in pathogenic Escherichia coli. Strains of various pathotypes were selected to establish the growth conditions for the formation of biofilms in the BioFlux 200 system on abiotic (glass) or biotic (eukaryotic cell) surfaces. Biofilm formation on glass was observed for the majority of strains when these were grown in M9 media at 30°C but not in RPMI at 37°C. In contrast, HRT-18 cell monolayers enhanced binding and, in most cases, biofilm formation of pathogenic E. coli in RPMI at 37°C. As a proof a principle to screen mutants, the ability of a diffuse adherent E. coli mutant strain lacking AIDA-1, a known mediator of attachment, was assessed in our models. When compared to the parental strain, the mutant formed a thin biofilm on glass or isolated clusters on an HRT-18 monolayer. In conclusion, we describe a microfluidic method for high throughput screening, which could be used to identify novel factors involved in E. coli biofilm formation and host-pathogen interactions under shear force.