In this study, the chitin of adult Mediterranean flour moth (Ephestia kuheniella) (Cht) was extracted and then converted to chitosan by deacetylation process to achieve the chitosan derived from E. kuheniella (Chs). The new chitosan-based scaffold was produced using the polyvinyl alcohol (PVA) co-electrospinning technique. The degree of deacetylation was obtained using the distillation-titration and Fourier transform infrared spectroscopy. The surface morphology and crystallinity index of Chs were observed using scanning electron microscopy and X-ray diffraction analysis, respectively, and compared with the commercial chitosan (Chs). Thermogravimetric analysis was used to estimate two chitosans' water content and thermal stability. The average molecular mass analysis was performed using viscometry. Moreover, the minimum inhibitory concentration and DPPH assay were used to study the antimicrobial activity and antioxidant potential of the Chs, respectively. Accordingly, Chs was smoother with fewer pores and flakes than Chs and its crystallinity index was higher than Chs. The water content and thermal stability were lower and similar for Chs compared to Chs. The average molecular mass of Chs was ~ 5.8 kDa, making it classified as low molecular weight chitosan. The antimicrobial activity of Chs against a representative Gram-negative bacteria; E. coli resulted to be the same as Chs. However, less effective than Chs against a representative Gram-positive bacteria is S. aureus. The Chs/PVA ratio scaffold was optimized at 30:70 to fabricate a uniform nanofiber scaffold.