New biomimetic micro- and nano-CsU-based fibrous scaffolds electrospun from solution containing high purity-medical grade chitosan (CsU) of fungus origin (CsU1, Mv ~174,000 and CsU2, 205,000, degree of deacetylation (DDA) ~65%) and polyethylene oxide (PEO, Mv ~ 900,000), in the presence of given amounts of Triton X-100 (from 0.01 to 0.5 wt%) as surfactant were fabricated. We demonstrate that by carefully selecting compositions and surfactant levels, porous mats with CsU content up to 90% (at this molecular weight and DDA) were achieved. Remarkable long-term stability in water or phosphate buffer solution storage were obtained by developing post-electrospinning treatment allowing the complete elimination of the PEO from the CsU-fibers as demonstrated by TGA, DSC and ESEM analysis. Subsequent reacetylation procedure was applied to convert 2D biomimetic chitosan mats to chitin (CsE)-based ones while preserving the nanofiber structure. This innovative procedure allows tuning and modifying the thermal, mechanical properties and more importantly the biodegradation abilities (fast enzymatic biodegradation in some cases and slower on the others) of the prepared nanofibrous mats. The established reproducible method offers the unique advantage to modulate the membrane properties leading to stable 2D biomimetic CsU and/or chitin (CsE) scaffolds tailor-made for specific purposes in the field of tissue engineering.