Antibacterial coatings based on bacterial cellulose (BC) have been widely used in many fields including food packaging and wound dressing. In this study, we aimed to synthesis of colloidal AgNPs and BC/ AgNP composite by using BC as a reducing and capping agent in one step reaction induced by gamma-ray. Bacterial strain Komagataeibacter rhaeticus N1 MW322708 was used for biosynthesis BC by inoculation on Hestrin and Schramm medium and incubated statically at 35 °C for 10 days. BC sheet was formed, harvested, purified, and dried, then used for the synthesis of AgNPs and BC/AgNP by soaked 0.05 g of dried BC in 10ml of 1mM aqueous AgNO3 solution for 2h and then irradiated by gamma-ray under different doses. Color change from yellow to deep brown indicated the synthesis of AgNPs and BC/AgNP. The optical spectra of synthesized AgNPs revealed that the surface plasmon resonance was localized around 420 nm. DLS analysis showed that the mean diameter of AgNPs was 49.5 nm with a -19.36-mV value of zeta potential. TEM images revealed the spherical shape of synthesized AgNPs. The results of FESEM, FTIR, and XRD confirmed the formation of BC/AgNO3 composite. The highly crystalline nature of the BC membrane and BC/AgNP composite was observed in XRD measurements with a crystal size of 5.416 and 5.409 nm, respectively. The antibacterial activity of BC and BC/AgNP against pathogenic bacterial isolated from Pastirma food samples revealed that BC does not show antibacterial activity, while BC/AgNP composite showed antibacterial potency against Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes, Proteus mirabilis, and Escherichia coli, with an inhibition zone of (mm) 9±1, 9±0.57, 10±1.15, 8±0.5 and 7±0.28, respectively. We concluded that this novel method presented in this paper offers a promising route for both AgNPs and BC/AgNP composites synthesis using a green, renewable biopolymer as a multifunctional agent and potential to be applied in the future development of food packing, biomedical instruments, and therapeutics.