This pilot train study compares the biofouling potential of two river waters that had been subjected to different pretreatment schemes conducting to equivalent microbial cells contents but different total and biodegradable organic carbon concentrations. Permeability and pressure drop of nanofiltration (NF) membranes were continuously monitored during the experiments. After a twenty week period of filtration, the membrane modules were autopsied, the composition and organization of membrane foulants were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and confocal laser scanning microscopy (CLSM). The ATR-FTIR spectra and CLSM observations of the fouled membranes revealed the presence of a biofilm at the membranes surface for the two feed waters. A positive correlation was observed between the biodegradable organic carbon concentration (BDOC) of feed water and the biofouling extent at the membrane surface: the BDOC concentration of feed water increase induced higher biofilm development. A longitudinal pressure drop increase was observed only with the water containing the higher concentration of BDOC whereas permeability decreases were equivalent with both feed waters. In conclusion, BDOC control through efficient pretreatments of raw water is a critical point for prevention of biofouling and maintenance of performances of NF membranes used in river water treatment.