Biphasic release bio-composite films of the low water-soluble drug, linezolid (LNZ), were formulated using the solvent casting technique. Different polymers and plasticizers (gelatin, Tween 80, polyethylene glycol 400, and glycerol) were assessed for the preparation of bio-composite films. An I-optimal design was applied for the optimization and to study the impact of polymer concentration (X), plasticizer concentration (X), polymer type (X), and plasticizer type (X) on different LNZ-loaded bio-composite films. The film thickness, moisture content, mechanical properties, swelling index, and percentage of drug release at fixed times opted as dependent variables. Results demonstrated a significant effect of all independent variables on the drug release from the prepared bio-composite films. The plasticizer concentration significantly increased the thickness, moisture content, elongation at break, swelling index, and drug release and significantly reduced the tensile strength. The optimized LNZ-loaded bio-composite film comprised of 15% Tween 80 and 30% PEG 400 was highly swellable, elastic, acceptable tensile properties, safe, maintained a moist environment, and indicated great antimicrobial activity against both (ATCC 25922) and methicillin-resistant (MRSA), which are common wound infectious bacteria. The present study concludes that the optimized LNZ-loaded bio-composite film was successfully designed with fast drug release kinetics and it could be regarded as a promising novel antimicrobial wound dressing formulation.