Biomass attachment and growth on high pressure membranes alter the surface characteristics and rejection performance of nanofiltration membranes. Along with electrostatic interaction and size exclusion, hydrophobic interaction between solutes and membrane surface play the major role in the separation process. Therefore, in attempt to properly quantify the surface energy of clean and biofouled membranes, different contact angle techniques were applied in this research. The surface energies of membranes were determined on dry, wet and hydrated surfaces. Results indicate that drying of the membrane surface leads to a modification of the surface properties, which are therefore not representative of the membrane in its operational conditions. Immersing the membrane in water resulted in detachment of biomass material into the surrounding liquid, thus hampering a correct estimation of the contact angle. Contact angle measurements on hydrated surfaces, on the contrary, produced reproducible results, which are consistent with current knowledge. In addition, when the values obtained by hydrated method were applied in a predictive model earlier developed, a significant improvement in predictions resulted.