Natural biopolymers are attracting the researcher's attention because of their magnificent properties and various biological activities. Among them, chitosan is the deacetylated form of chitin which is present in the fungal cell wall. In the present study, chitosan was extracted from Aspergillus niger on malt yeast broth (MYB) with yield 8%, degree of acetylation 69.5 % and molecular weight 2223.11 Da. Nano-chitosan was prepared through ionic gelation method then characterized through dynamic light scattering which revealed that the zeta potential was 37 mV and polydispersity index (PDI) reached 0.48 while transmission electron microscope (TEM) showed the spherically shaped prepared nanoparticles had particle size ranged from 1.77 to 8.17 nm. Thermoplastic Polyurethane (TPU) fibers loaded with chitosan and nano-chitosan were synthesized through electrospinning then morphologically characterized by scanning electron microscope (SEM) showing that chitosan loaded TPU fibers were comparatively rougher than that of nano-chitosan loaded TPU fibers which were quite smooth. The Limiting oxygen index (LOI) value of chitosan loaded TPU fibers was only 20.8% and the UL-94 resulted in V-1. However, the LOI value of nano-chitosan loaded TPU fibers increased up to 26.6% and the UL-94 result achieved V-0 rating. Moreover, the tensile strength of the chitosan loaded TPU fibers was 12.3 ± 0.2 MPa, while the nano-chitosan loaded TPU fibers was 17.9 ± 0.5 MPa. Finally, nano-chitosan loaded TPU fibers showed the maximum antibacterial activity with inhibition zone diameter reached 40 mm and MIC value 15.6 µg/mL.