Bionanocomposites with different loadings of silver (Ag) were prepared via synthesis of Ag nanoparticles (AgNPs) using the wet chemical reduction method in the lamellar space layer of the organo-sepiolite/chitosan (O-SEP/CS). The prepared O-SEP/CS/Ag bionanocomposites were characterized using various analysis methods for their structure, morphology, and optical properties. The characteristic absorption bands from the UV-visible absorption spectrum confirmed the formation of AgNPs. The antibacterial activities of O-SEP/CS/Ag bionanocomposites were investigated against gram-positive and gram-negative bacteria using the disc diffusion method. The results suggest that O-SEP/CS/Ag bionanocomposites can be useful in wide range of bio-medical applications because of high antibacterial activity.