A sensitive and simple method based on ultrasonication extraction with a hexane/acetone (2:1, v/v) mixture, followed by clean up of the extract by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS) detection, has been developed and validated for the analysis of 20 estrogenic endocrine-disrupting chemicals (EEDCs) including phenolic xenoestrogens, synthetic and natural estrogens in river sediment. After extraction and purification, analytes are derivatised with a BSTFA/TMCS/pyridine (49:1:50, v/v/v) mixture and quantified by GC/MS. The GC/MS method involves switching between electron ionisation (EI) and chemical ionisation (CI); it also switches between selected ion storage and tandem mass spectrometry detection. The applicability of the method has been demonstrated by analysing extracts of French river sediments for which bioanalytical tests (in vitro) had already shown that they were impacted by estrogenic endocrine disrupters. The biological contribution of all the products detected in each sediment extract was compared to the estrogenic activity measured by bioassays.