Bacterial cellulose (BC) is used in various fields for its unique physical properties, but does not have the antimicrobial properties needed for the food and biomedical industries. Co-culture fermentation is a method commonly used in biotechnology to address high costs. A nisin-containing BC film (BC-N) was obtained by co-cultivating the BC-producing strain Enterobacter sp. FY-07 with the nisin-producing strain Lactococcus lactis N8. The physical properties of BC-N were similar those of BC, but the BC-N film had a specific strong inhibitory effect on Gram-positive bacteria. The antibacterial mechanism of BC-N was pore formation, but the obtained BC-N film had no significant impact on mammalian cell viability. This study provides a low-cost, facile and efficient technique to confer BC with antimicrobial properties. This strategy can be applied to introduce other functions into BC, and develop applications for BC polymers.