We examined the applicability of environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM) for non-invasive imaging of intact bacterial biofilms. Fully hydrated images of Pseudomonas aeruginosa biofilms developing on the silicon disks in an in vitro experimental model were detected simply but clearly by ESEM and CLSM. Three-dimensional spatial images of the bioflims were obtained by ESEM using specimens with neither drying nor conductive coating. Horizontal and vertical sectioning images of the biofilms were obtained by using CLSM in conjunction with positive fluorescence staining techniques; acridine orange and sufranin were successfully used for detection of bacterial cells in the biofilms, while extracellular polymeric substances were obtained selectively with FITC-concanavalin A. The new technology reported here is applicable to diagnostic study as well as fundamental study of biofilm-associated infections in the urinary tract.