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The goal of this study is to develop a high frequency, pulse-echo ultrasound system to non-invasively image and characterize biofilms in children during nasopharyngeal (NP) colonization with potential otopathogens and during acute ear infections (acute otitis media, AOM). This study focuses on biofilms grown in vitro on silastic substrate to determine the feasibility of detecting and characterizing parameters such as biofilm thickness, backscatter coefficient, integrated backscatter, shift of center frequency and reduction of bandwidth. These parameters are needed to understand image properties and design an efficient non-invasive protocol to identify biofilms, map their progression over time, and differentiate between singlespecies and multiple-species biofilms. This paper uses a 15MHz focused, immersion transducer in a systematic study investigating the feasibility of high frequency ultrasound as an in vivo biofilm imaging technology and therapeutic tool.