This study considers the effect of cross-flow velocity and transmembrane pressure on flux and rejection behavior during nanofiltration of Eurasian water milfoil hydrolysate. Fractionation by nanofiltration was applied to obtain a glucose-rich permeate solution and a concentrated retentate solution of other sugars and phenolics. Nanofiltration was performed in a cross-flow rectangular flat-sheet cell (MaxiMem, Prozesstechnik GmbH). Membrane Microdyn Nadir (TM) NP030 with molecular weight cut-off 500 Da was used. The velocity and shear stress distribution on the membrane surface was modelled by CFD simulations. Mild hydrodynamic conditions (cross-flow velocity and pressure) are recommended based on the observed permeate flux and rejection difference between the targeted groups of compounds. The results are discussed in rapport with existing experimental observations in the literature. (C) 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.