The biofilm is a community of microorganisms found adhered to a surface, growing in an exopolysaccharide matrix whose composition varies between bacterial species. It has been reported that chronic infections are closely related to the formation of biofilm; it represents a defense mechanism in which bacterial cells survive the bacterial stress, to host immune response and even resist some antimicrobial agents. The calcofluor white is a fluorophore that binds to glycosidic bonds β(1-3) and β (1-4), generally used for identifying various species of fungi. This paper shows an assay to the detection of uropathogenic E. coli biofilm using the calcofluor staining.