Abstract: Antimicrobial cellulose nanocomposite films with in situ synthesized bimetallic nanoparticles were developed by a three-step process. These antimicrobial cellulose nanocomposite films with in situ synthesized bimetallic, silver (Ag) and copper (Cu) nanoparticles (NPs) through the apozem of medicinal important plant Vitex negundo (Vavili) leaves, and which is acts as a bio-reductant and stabilizing agent. Through a three-step process, the cellulose films were prepared via a solution casting method and loaded with the apozem of V. negundo (Vavili) leaves via the swelling process. The resulted nanocomposite films were characterized by XRD, UV, FTIR, SEM/EDAX, TGA, UTM and antibacterial measurements. The XRD studies confirm that the presence of AgNPs and CuNPs in the cellulose matrix. The formation Ag/Cu, bimetallic nanoparticles were observed by the change of the color of the cellulose films. The presence of UV absorption peaks at around ~ 415 (Ag) and ~ 570 nm (Cu) confirms the formation of bimetallic cellulose nanocomposite films. The SEM and EDAX spectrum confirmed the presence of Ag, Cu, and Ag/Cu elements. The average size of the Ag–Cu, Ag, and Cu nanoparticles generated were ~ 60, ~ 67, and ~ 74 nm, respectively. The UTM and TGA studies revealed that bimetallic nanocomposites have high strength and stability than monometallic nanocomposite films. The nanocomposites have shown good antibacterial activities towards gram-positive and gram-negative bacteria. These cellulose nanocomposite films stand as good candidature for food, medical, and disinfection packaging materials. Graphic Abstract: [Figure not available: see fulltext.]