The objective of this study is to investigate the morphological (scanning electron microscopicy images), thermal (differential scanning calorimetry), and electrical (conductivity) properties and to carry out compositional analysis (Fourier-transform infrared) of produced nonwoven fibrous materials adapted in biomedical applications as scaffolds. The orientation of produced nanofilaments was also investigated because it is considered as one of the essential features of a perfect tissue scaffold. Viscosity and electrical conductivity of solutions, used in the manufacturing process, were also disassembled because these properties highly influence the morphological properties of produced nanofibers. The nanofibrous scaffolds were fabricated via conventional electrospinning technique from biopolymer, synthetic polymer, and their blends. The chitosan (CS) was chosen as biopolymer and polyethylene oxide (PEO) of low molecular weight as synthetic polymer. Solutions from pure CS were unspinnable: beads instead of nanofibers were formed via spinning. The fabrication of pure PEO nanomats from solutions of 10 wt%, 15 wt%, and 20 wt% concentrations (in distilled water) turned out to be successful. The blending of composed CS solutions with PEO ones in ratios of 1:1 optimized the parameters of electrospinning process and provided the opportunity to fabricate CS/PEO blends nanofibers. The concentration of acetic acid (AA) used to dissolve CS finely spuninned the nanofibers from blended solutions and influenced the rate of crystallization of manufactured fiber mats. The concentration of PEO in solutions as well as viscosity of solutions also influenced the diameter and orientation of formed nanofibers. The beadless, highly oriented, and defect-free nanofibers from CS/PEO solutions with the highest concentration of PEO were successfully electrospinned. By varying the concentrations of AA and low molecular weight PEO, it is possible to fabricate beadless and highly oriented nanofiber scaffolds, which freely can found a place in medical applications.