This study aims to identify the nature of organic species (soluble or colloidal) governing membrane fouling within the context of a membrane bioreactor (MBR). A knowledge of their nature and origin (excreted during treatment or associated with the influent) can help to better adapt operational conditions and cleaning procedures. In this work, a fractionation step by ultracentrifugation and innovative organic matter characterisation were done in parallel with dead end microfiltration tests. The complementarities of classical parameters and innovative analyses can help depict foulant species physicochemical properties. Results show that the fouling layer is mostly governed by the deposition of soluble compounds whereas the impact of the colloidal fraction (poorly presents in supernatant) is less. HPLC-SEC and fluorescence analyses reveal the important role of proteins-like substances (polypeptides) in membrane bioreactor fouling.