Hydroacoustics is a common sampling tool in freshwater and marine ecosystems, yet the full potential of this tool remains restricted, owing to an incomplete understanding of the influence of many data collection settings. In particular, investigation into the effects of the pulse duration setting on common hydroacoustic measures remains limited. Hydroacoustic theory suggests that pulse duration can affect how many single targets (e.g., individual fish or invertebrates) are detected, the mean target strength (TS) of sample cells, and estimates of target density, especially in ecosystems with an abundance of potential targets. To quantify the influence of the pulse duration setting on these important and commonly used hydroacoustic measures, we sampled multiple ecosystems (Lake Erie and three tributary reservoirs) that span a wide gradient in fish density and regularly use hydroacoustics to assess fish biomass.