Targeted ultrasound contrast agents (UCA) offer a novel, potential method for diagnostic imaging of biofilm infections. Currently, there is no established method for molecular imaging of in vitro biofilm infections with any type of modality. For infective endocarditis, the case of biofilm formation on damaged or diseased heart valves, an early diagnostic method might greatly reduce the associated high mortality rate. Fluorescently labeled lectins were used with targeted ultrasound contrast agents in conjunction with epifluorescence microscopy and time-resolved scanning acoustic microscopy for visualization and characterization of the extracellular polymeric substances (EPS) of two types of infectious biofilms (Staphylococcus aureus and Pseudomonas aeruginosa). Noninvasive acoustic measurements of the biofilms were conducted at high-frequency (100 MHz) with a time-resolved scanning acoustic microscope. Measurements of the reflection and transmission coefficients, over a broad frequency spectrum for a range of biofilm growth stages in both, the presence and absence of targeted UCA were obtained. The biofilm morphology and structure were also investigated with acoustic microscopy as a function of growth. [This work was supported by the National Institutes of Health Grants NIH 2 P20 RR016453-05A1 and NIH 2 G12 RR0030161-21.].