Aims: This study evaluated the effect of protozoan movement and grazing on the topography of a dual-bacterial biofilm using both conventional light microscopy and a new ultrasonic technique. Methods and Results: Coupons of dialysis membrane were incubated in Chalkley's medium for 3 days at 23°C in the presence of bacteria (Pseudomonas aeruginosa and Klebsiella aerogenes) alone, or in co-culture with the flagellate Bodo designis, the ciliate Tetrahymena pyriformis or the amoeba Acanthamoeba castellanii. Amoebic presence resulted in a confluent biofilm similar to the bacteria-only biofilm while the flagellate and ciliate created more diverse biofilm topographies comprising bacterial microcolonies and cavities. Conclusions: The four distinct biofilm topographies were successfully discerned with ultrasonic imaging and the method yielded information similar to that obtained with conventional light microscopy. Significance and Impact of the Study: Ultrasonic imaging provides a potential way forward in the development of a portable, nondestructive technique for profiling the topography of biofilms in situ, which might aid in the future management of biofouling.