The biochemical characterization of Extracellular Polymeric Substances (EPS) excreted in a European intertidal mudflat (Marennes–Oléron Bay) was performed. Experiments were carried out for the first time in situ, by using an improved extraction recently developed. This innovative procedure, using a cation exchange resin (Dowex), allows separating precisely different fractions of EPS, especially pure bound EPS. Moreover, it avoids the contamination of EPS fractions by residual and intracellular polymers, enabling to properly estimate polymeric contents in each fraction. The results were partly similar to conventional results described in the literature and the amount of colloidal carbohydrates (146 μg/g of dry sediment) extracted by the Dowex method fitted well with different EPS estimation in European mudflats. Colloidal carbohydrates were essentially composed of glucose (>50%), a carbon source rapidly consumed by the various communities in the sediment. Pure bound carbohydrates were composed of specific carbohydrates (28% rhamnose, 22% xylose). Residual fractions, considered as containing some refractory bound EPS and mostly other internal polymeric substances, presented a more varied composition rich in carbohydrates: galacturonic acid (20%), mannose (19.5%), glucose (19%), arabinose (15%), xylose (8%), galactose (7%).Research highlights► Results confirmed the relevance and the effectiveness of Takahashi’s method for in situ experiments, ► Bound fractions were biochemically different from the two other fractions, thanks to the presence of large amounts of deoxy sugars and uronic acids, ► Pure bound carbohydrates were composed of specific carbohydrates (28% rhamnose, 22% xylose), ► The surprising lack of fucose has been correlated to the sampling period, ► The presence of inositol was detected for a first time in situ.