Noninvasive measurements over a biofilm, a three-dimensional (3-D) community of microorganisms immobilized at a substratum, were made using an acoustic microscope operating at frequencies up to 70 MHz. The microscope scanned a 2.5-mm by 2.5-mm region of a living biofilm having a nominal thickness of 100 microm. Spatial variation of surface heterogeneity, thickness, interior structure, and biomass were estimated. Thickness was estimated as the product of the speed of sound of the medium and the interim between the highest signal peak and that of the substratum plane without biofilm. The thickest portions of biofilm were 145 microm; however, slender structures attributed as streamers extended above, with one obtaining a 274-microm height above the substratum. Three-dimensional iso-contours of amplitude were used to estimate the internal structure of the biofilm. Backscatter amplitude was examined at five zones of increasing height from the substratum to examine biomass distribution. Ultrasound-based estimates of thickness were corroborated with optical microscopy. The experimental acoustic and optical systems, methods used to estimate biofilm properties, and potential applications for the resulting data are discussed.