Randomly oriented fiber mats of chitosan–polyethylene oxide matrix reinforced with cellulose nanocrystals (CNCs) were prepared by electrospinning technique. The cellulose nanocrystals used were isolated using hydrochloric acid (CNCHCl) or sulphuric acid (\({\text{CNC}}_{{{\text{H}}_{ 2} {\text{SO}}_{ 4} }}\)) and the concentration of CNCs was 50 wt% in the electrospun mats. The surface characteristics of the nanocrystals were found to affect the dispersion, viscosity, conductivity and zeta-potential of the respective spinning solutions and resulted in better spinnability, homogeneity as well as crosslinking of CNCHCl based nanocomposite fiber mats compared to \({\text{CNC}}_{{{\text{H}}_{ 2} {\text{SO}}_{ 4} }}\) ones. The microscopy studies showed that the diameter of the electrospun fibers decreased with the inclusion of both types of nanocrystals and that crosslinking decreased the porosity of the mats. The tensile strength and tensile modulus of the mats increased with the addition of nanocrystals and increased further for the CNCHCl based mats (58 MPa, 3.1 GPa) after crosslinking. The as-spun CNCHCl based mats had average pore diameters of 1.6 μm and porosity of 38 %. The water vapor permeability and the O2/CO2 transmission increased with the addition of CNCHCl. The used nanocrystals as well as electrospun mats showed non-cytotoxic impact on adipose derived stem cells (ASCs), which was considered favorable for wound dressing.