Random and highly aligned bead-free chitosan nanofibers (NFs) were successfully prepared via electrospinning by keeping the applied voltage (22 kV), flow rate (0.4 mL h−1), needle diameter (0.8 mm), and needle to collector distance (100 mm) constant while varying the solution concentration and collector rotation speed. No electrospinning was observed for lower solution concentrations, i.e., 1–3 wt% (w/v), whereas a decrease in the number and size of beads and microspheres, and bead-free NFs were obtained when the concentration of solution was increased from 4 to 6 wt%.