Due to the fundamental physiology and current limits in detection methods, the majority of microorganisms, so-called “unseen majority (USM)”, in the natural world are not accessible using traditional microbiological tools. Small USM bacteria are dominant and have significant ecological functions in most aquatic environments, where nutrient concentrations are low. As they are sensitive to conventional high nutrient media and have small bio-volumes (less than 0.1 μm3), small bacteria often escape traditional cultivation attempts and are therefore still poorly described. In this review, the concepts relating to the characterization of small cells are compared. We also review the cultivation and detection methods and the distribution of small bacteria in aquatic environments, followed by in-depth discussion of the ecological functions of small bacteria. Finally, the future prospects of USM bacteria are discussed in terms of their growth physiology and applications in water quality evaluation.