In this present work, fungal nanochitosans, with very interesting particle size distribution of 22 µm, were efficiently generated in high-yield production using a high-pressure water jet system (Star Burst System, Sugino, Japan) after 10 passes of mechanical treatment under high pressure. The specific characterization of fungal chitosan nanofibers suspensions in water revealed a high viscosity of 1450 mPa.s and an estimated transparency of 43.5% after 10 passes of fibrillation mechanical treatment. The mechanical characterization of fungal nanochitosan (NC) film are very interesting for medical applications with a Young’s modulus (E), a tensile strength (TS), and elongation at break (e%) estimated at 2950 MPa, 50.5 MPa, and 5.5%, respectively. Furthermore, we exhibited that the fungal nanochitosan (NC) film presented very good long-term antioxidant effect (reached 82.4% after 96 h of contact with DPPH radical solution) and very interesting antimicrobial activity when the nanochitosan (NC) fibers are mainly activated as NC-NH3+ form at the surface of the film with 45% reduction and 75% reduction observed for S. aureus (Gram-positive) and E. coli (Gram-negative), respectively, after 6 h of treatment. These promising antimicrobial and antioxidant activities indicated the high potential of valorization toward biomedical applications.