The present study investigated that chitosan production of Rhizopus oryzae NRRL 1526 and Aspergillus niger ATCC 16404. Fungal chitosans were characterized by scanning electron microscopy (SEM)-energy dispersive X-ray analysis, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimeter and deacetylation degrees of fungal chitosans were determined. The percentage yield of Ro-chitosan and An-chitosan were determined as 18.6% and 12.5%, respectively. According to percentage of chitosan yield and the results of the characterization studies, chitosan that obtained from Rhizopus oryzae NRRL 1526 was selected for subsequent studies. Cytotoxicity of chitosan obtained from Rhizopus oryzae NRRL 1526 was determined by MTT assay on human dermal fibroblast cell line. Acording to results of the cytotoxicity test fungal chitosan was nontoxic on cells. The high cell viability was observed 375 μg/mL concentration at 24th, 48th h periods and at the 187.5 μg/ml 72nd h periods on cells. The fungal chitosan obtained from Rhizopus oryzae NRRL 1526 was used to fabrication of electrospun nanofibers. Fungal chitosan based polymer solutions were prepared by adding different substances and different electrostatic spinning parameters were used to obtain most suitable nanofiber structure. Characterization studies of nanofibers were carried out by SEM, FTIR and X-ray diffraction. The most suitable nanofiber structure was determined as F4 formula. The nanofiber structure was evaluated to be thin, bead-free, uniform, flexible and easily remove from surface and taking the shape of the area. After the characterization analysis of fungal chitosan it was determined that the chitosan, which obtained from Rhizopus oryzae NRRL 1526 is actually chitosan polymer and this polymer is usable for pharmaceutical areas and biotechnological applications. The electrospun nanofiber that blends fungal chitosan and PCL polymers were fabricated successfully and that it can be used as fabrication wound dressing models. RESEARCH HIGHLIGHTS: Extraction of chitosan from Rhizopus oryzae NRRL 1526 and Aspergillus niger ATCC 16404 and characterization scanning electron microscopy-energy dispersive X-ray analysis, Fourier transform infrared spectroscopy, differential scanning calorimeter. Fabrication and characterization of the fungal chitosan/PCL electrospun nanofibers.