In fisheries acoustics, the target strength (TS; dB re m2) is used to compute biological metrics such as fish biomass and density. The TS is challenging to characterize because of its stochastic relationship with fish physiology, orientation, depth, species assemblage, and size distributions. These challenges were addressed by using acoustic and physical samples of fish from trawls in the Penobscot River Estuary, Maine. The pelagic species assemblage was dominated by clupeids and osmerids. The TS was measured from individual fish using single target detection and echo tracking algorithms. An expectation-maximization algorithm was applied to identify the components of the TS and total length (TL; cm) distributions for the mixed species assemblages. Regressions were used to estimate the parameters of TS = α log10(TL) + β. The parameters, α = 31.2 [standard error (SE) 0.87] and β = -79.6 (SE 0.93), were similar to published studies from these species, but our slope and intercept were higher than those in studies from freshwater and lower than those from marine systems. These results suggest that acoustic surveys in estuaries with mixed species assemblages should carefully consider alternatives to "standard" TS-fish length equations. These results will provide necessary parameters to allow for interpretation of acoustic survey data from systems with a similar composition of pelagic species.