Compounds displaying estrogenic potential can lead to serious negative aquatic ecosystem impacts, and exposure to mixtures of endocrine disruptors present in environmental matrices with very complex characteristics, including sediments, can cause serious risks to biota, animals, and humans. Within this perspective, the aim of the present study was to assess estrogenic activity and toxicity in sediments sampled from Guanabara Bay, the most important, although chronically polluted, estuary in southeastern Brazil. To this end, estrogenic activity determinations were performed by the yeast estrogen screen (YES) assay, and acute toxicity assays were carried out employing Vibrio fischeri. Estrogenic activity was quantified up for 8.7 ng g−1 in E2‐EQ, although high levels of cytotoxicity (up to 95%) were observed and might have hindered the determination of the real estrogenic potential in most samples. Acute toxicity to V. fischeri ranged from 0 to 45.21%. These results point to potential deleterious effects caused by micropollutants and underline the urgency of continuous monitoring of the degradation degree of the Guanabara Bay estuary.