Multimetric fish-based indices have been increasingly gaining importance in Europe, as the Water Framework Directive (WFD) requires fish fauna, and particularly its composition and abundance, to be taken into account in the assessment of the ecological quality of continental surface waters, including transitional waters. These indices are composed of several metrics, mostly related with structural and functional characteristics of fish communities, such as species richness, the role of nursery areas, or trophic web structure. Therefore, ecological quality assessments should ensure that these structural and functional characteristics of fish communities were covered by the sampling methods used. In the present work, the influence of sampling effort on several metrics of the Estuarine Fish Assessment Index (EFAI) was studied. Pseudo-random samples were generated from data of four Portuguese estuaries and bootstrap cycles were performed, in order to obtain metrics’ means and standard deviations per number of hauls analysed. The number of hauls necessary for the means to level off differed with the metrics considered. Generally, for metrics on percentages (percentage of marine migrants, percentage of estuarine residents and percentage of piscivores) the curve levelled off with less than 20 hauls, both for the estuary as a whole and for different estuarine salinity zones. On the other hand, metrics on species richness required much larger samples. In order to decrease to −5% the current estimated bias of metrics, the WFD sampling costs would have to be more than 3 times higher than they currently are. The findings in the present study are of great importance for an effective assessment of estuarine ecological quality and particularly in the context of the WFD, as the metrics studied are common to other Member State indices.