We investigated the activity of the multixenobiotic resistance (MXR) phenotype, a biological defense system in aquatic organisms, in the fish assemblages of two tropical estuaries with different degrees of environmental impacts, the Paraiba River and Mamanguape River Estuaries. The aim of this work was to compare the activity of the MXR phenotype of different fishes to test the hypothesis that each species has an inherent activity level and to use this activity as a bioindicator of aquatic contamination. We assessed the MXR activity of the gills, using rhodamine B (RB) accumulation assay. The results demonstrated a species-specific difference in the MXR activity of fishes caught in the same estuarine zone. Also, the pelagic species Eucinostomus melanopterus, Eucinostomus argenteus, and Lutjanus jocu had higher RB accumulation, while the demersal species Sphoeroides testudineus and Sphoeroides greeleyi had the lowest RB accumulation, suggesting that the ecological characteristic of fish in the water column exerts an influence on MXR activity. Besides, we demonstrated the potential of using the gill MXR activity of the key estuarine species, the Brazilian silversides Atherinella brasiliensis, as a tool for biomonitoring estuaries. K E Y W O R D S biomarker, Brazilian silverside, P-glycoprotein, MXR, rhodamine B, tropical estuaries SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Macêdo AKS, da Silva JRP, dos Santos HB, Thomé RG, Vendel AL, Amado EM. Estuarine fish assemblages present a species-specific difference in the multixenobiotics resistance activity. J. Exp. Zool. 2019;331: 530-539. https://doi.