To synthesize biomaterials for antibacterial applications, chitosan (CS) and chitooligosaccharide (COS) were successfully incorporated into bacterial cellulose (BC) matrix respectively. The representative Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) were selected to evaluate the antibacterial effects of the composite membranes. The results manifested BC-CS and BC-COS membranes exhibited remarkable antibacterial properties against both bacteria with the inhibition ratio of 99.99 ± 0.01% (S. aureus) and 99.99 ± 0.01% (E. coli) for BC-CS, 99.64 ± 0.18% (S. aureus) and 90.56 ± 0.06% (E. coli) for BC-COS. The morphology obtained by scanning electron microscopy (SEM) showed BC-CS and BC-COS composite membranes possessed lower porosity and denser structure in comparison to pure BC. Moreover, in contrast with BC-CS, BC-COS performed better mechanical properties and also displayed stronger ABTS radical scavenging capacity of 92.62 ± 2.85%. These results suggested that the development of BC-COS composite membrane would open up the possibility for the antibacterial applications in food and medical fields.