Soluble microbial products are one of the major fouling agents in membrane bioreactor (MBR). It is accepted that high molecular weights polysaccharides are the main contributors to membrane fouling but the presence in bulk solution of proteins and humic-like substances make fouling layer more complex. To better understand the role of both components in fouling establishment, they were quantified and characterized in bioreactor and permeate under various operating conditions (sludge retention time (SRT), synthetic or real wastewater (SWW or RWW), rapid variation of food to microorganisms (F/M) ratio). With SWW at hydraulic retention time (HRT) of 24 h, a larger part of proteins possessing small molecular weights (< 1 kDa) were obtained with increasing SRT from 20 to 50 d. At 50 d, these proteins present better retention (93%) and could participate in lowering gel layer porosity. MBR operating at SRT of 20 d was then preferable. At respective SRT and HRT of 50 d and 24 h with SWW, F/M ratio decrease (from 0.2 to 0.1 kg (COD).kg(MLVSS)(-1).d(-1) during 24 h) provoked implementation of a compact fouling layer which provoked a high TMP increase (0.83 kPa.h(-1)). Biodegradation of proteins involved in bio clusters structures were implied in this phenomenon.