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One of the main limitations of membrane applications for water treatment is biofouling, which causes a decrease of membrane performances and is often very difficult to remove by chemical cleanings. To act preventively, membrane pretreatment has to be optimized to minimize the biofouling potential of the feedwater. This study compares different pretreatment processes implemented upstream from nanofiltration (NF), and their ability to reduce of biofouling on NF elements. Two identical one-stage NF membrane pilots were fed different water qualities from two water treatment plants in France (Neuilly-sur-Marne and Choisy-le-Roi) during several long filtration tests. Both pilots included a pretreatment step made of pH neutralization, 20 and 6 µm cartridge filtration (except during test 4), and antiscalant injection. UV disinfection was realized by a pilot reactor containing a low pressure UV lamp operated at 400 J/m<sup>2</sup>. At the end of every test, NF spiral-wound modules were autopsied and analyzed. The fouling deposit was characterized by ATR-FTIR spectroscopy and by epifluorescence microscopy (DAPI staining for total bacteria counts, SYTO9-propidium iodide for live/dead distinction, FTIC and TRITC-conjugated lectins for exopolysaccharides visualization). The deposit dry weight, wettability, ATP and proteins contents were also determined. This study highlights the importance of pretreatment to control biofouling on NF membranes. Includes 17 references, tables, figures.