In this study cellulose-rich membranes were fabricated from untreated and treated hardwood biomass solutions in 1-ethyl-3-methylimidazolium acetate ([Emim][OAc])—dimetylsulfoxide (DMSO) system via wet phase separation. Wood treatment methods aimed to get purified cellulose fraction of wood. Treatment sequence was as followed: deep eutectic solvent pretreatment, sodium chlorite bleaching, and alkaline treatment. Resulted biomass after each treatment step was characterized by chemical composition and crystalline fraction content. Flat-sheet membranes were produced from biomass samples after each treatment step. Characterization of membranes included measurements of pure water permeability and (poly)ethyleneglycol 35 kDa retention, Fourier-transform infrared and Raman spectroscopy, X-ray diffraction measurements and thermogravimetric analysis. The study revealed that it was possible to fabricate membrane from untreated wood as well as from wood biomass after each of treatment steps. The resulted membranes differed in chemical composition and filtration performance. Membrane prepared directly from untreated wood had the highest permeability, the lowest retention; and the most complex chemical composition among others. As treatment steps removed lignin and hemicelluloses from the wood biomass, the corresponding membranes became chemically more homogeneous and showed increased retention and decreased permeability values.