As part of an intensive, multidisciplinary limnological study of Lake Biwa, Japan during 23 August through 15 September 1993, multifrequency acoustic backscatter profiles of small fish were collected at a single location. Estimates of volume scattering strength using calibrated inverted echo sounders at five frequencies (29–198 kHz) were combined with echo statistics and simple scattering models to estimate approximate sizes and population densities. Echo amplitude probability density functions (PDFs) were used to constrain population density estimates in two ways: (1) PDF shape estimates were used to find the critical density among four different beam-width sonars, and (2) an isolated echo convolution model was fit to measured PDFs from the 198-kHz sonar, yielding the scattering cross-section for individual fish. An epilimnion scattering layer was consistently found, bounded below by the 24 °C isotherm at 10- to 15-m depth, likely consisting of juvenile Hasu (Opsariichthys uncirostris) of length 35 to 45 mm, with acoustic cross-sections of 4–9×10−5 m2 (target strengths of −55 to −51 dB re: 1 m2), and mean population densities in the range of 0.2–0.5 per m3. Individuals or small schools of adult fish (likely O. uncirostris, Plecoglossus altivelis, and/or Gnathopogon caerulescens) with typical lengths of 10 to 20 cm were intermittently observed at or above the main thermocline with instantaneous densities of 1 to 25 per m3. The typical depth of occurrence of these adult fish varied from 2.8 m during the day to 15.2 m at night.