Today, hydroacoustic technologies are able to monitor fish populations and assess fish biomass by providing data of a high spatio-temporal resolution, and without damaging the environment. Such techniques are widely used in lakes and marine environments, but they remain much less tested in shallow waters - especially in estuarine habitats. Our study was carried out using a strictly methodological approach. This was necessary in order to take into account the highly changeable nature of the ecosystem in question. Hydroacoustic surveys were conducted in the mesohaline part of the Gironde estuary using SIMRAD EK60 split-beam echosounders, operating simultaneously at two frequencies (70 kHz and 120 kHz). Different mobile hydroacoustic surveys were tested in different seasons, with vertical acquisition, in order to compare variability of results due to the sampling strategy, and to validate the relevance of our design by repeating the surveys. The results obtained on survey design choices gave similar outcomes when sampling was carried out with or against the current and between two mobile designs. The comparison between flood and ebb tides gave different results from one day to another but we observed the same evolution for each flood tide or for each ebb tide on several successive days. This acoustic study in the Gironde estuary was used to validate a reliable sampling mobile design. It is the first step in the process of gaining a better understanding of estuarine ecosystems, an understanding that will be enhanced by combining our data with data from a variety of other sources.