Functional wound dressing with tailored physicochemical and biological properties is vital for diabetic foot ulcer (DFU) treatment. Our main objective in the current study was to fabricate Cellulose Acetate/Gelatin (CA/Gel) electrospun mat loaded with berberine (Beri) as the DFU-specific wound dressing. The wound healing efficacy of the fabricated dressings was evaluated in streptozotocin-induced diabetic rats. The results demonstrated an average nanofiber diameter of 502 ± 150 nm, and the tensile strength, contact angle, porosity, water vapor permeability and water uptake ratio of CA/Gel nanofibers were around 2.83 ± 0.08 MPa, 58.07 ± 2.35°, 78.17 ± 1.04%, 11.23 ± 1.05 mg/cm2/hr, and 12.78 ± 0.32%, respectively, while these values for CA/Gel/Beri nanofibers were 2.69 ± 0.05 MPa, 56.93 ± 1°, 76.17 ± 0.76%, 10.17 ± 0.21 mg/cm2/hr, and 14.37 ± 0.42%, respectively. The antibacterial evaluations demonstrated that the dressings exhibited potent antibacterial activity. The collagen density of 88.8 ± 6.7% and the angiogenesis score of 19.8 ± 3.8 obtained in the animal studies indicate a proper wound healing. These findings implied that the incorporation of berberine did not compromise the physical properties of dressing, while improving the biological activities. In conclusion, our results indicated that the prepared mat is a proper wound dressing for DFU management and treatment.