© 2022In recent years, there is an increasing attention on biocompatible electrospun nanofibers for drug delivery applications since they provide high surface area, controlled and sustained drug release, and they mimic the extracellular matrix. In the present study, tetracycline hydrochloride (TCH) antibiotic loaded poly(ω-pentadecalactone-co-ε-caprolactone)/gelatin/chitosan nanofibrous membranes were fabricated as a controlled drug delivery system. Poly(ω-pentadecalactone-co-ε-caprolactone) copolymer has been enzymatically synthesized in previous studies, thus it provides an originality to the membrane. Combination of a synthetic polymer, a protein, and a polysaccharide in order to obtain a synergetic effect is another novelty of this work and there exists limited examples for such electrospun membrane. Varied amounts of TCH was electrospun together with poly(ω-pentadecalactone-co-ε-caprolactone)/gelatin/chitosan (50/40/10 vol ratio) polymer blend (fiber diameters ranged between 85.7–225.2 nm) and several characterizations (morphological and molecular structure, wettability characteristics, and thermal behavior) were applied to examine the drug incorporation. Subsequently, in vitro drug release studies were conducted and mathematical modeling was applied for the detection of transport mechanism of drug. TCH release proceeded 14 days through an initial burst release in first hour and followed by a sustained release. 1% TCH-loaded sample was shown as optimal preparation with 96.5% total drug release and 11.8% initial burst release. TCH-loaded preparations demonstrated a good antibacterial activity against Gram-positive (Staphylococcus aureus and Bacillus subtilis) bacteria and a limited effect (no inhibition zone observed below 3% TCH concentration) against Gram-negative (Escherichia coli) bacterium. Thus, TCH concentrations of ≥ 3% could be preferred to obtain a wide-spectrum effectiveness. The presented drug delivery system is suggested to be applied for treatment of skin infections as a wound dressing device.