Bacterial cellulose (BC) has received considerable attention due to its unique properties, including an ultrafine network structure with high purity, mechanical strength, inherent biodegradability, biocompatibility, high water-holding capacity and high crystallinity. These properties allow BC to be used in biomedical and industrial applications, such as medical product. This research investigated the production of BC by Gluconacetobacter hansenii ATCC 23769 using different carbon sources (glucose, mannitol, sucrose and xylose) at two different concentrations (25 and 50 g∙L−1). The BC produced was used to develop a biocomposite with montmorillonite (MMT), a clay mineral that possesses interesting characteristics for enhancing BC physical-chemical properties, at 0.5, 1, 2 and 3% concentrations. The resulting biocomposites were characterized in terms of their physical and barrier properties, morphologies, water-uptake capacities, and thermal stabilities. Our results show that bacteria presented higher BC yields in media with higher glucose concentrations (50 g∙L−1) after a 14-day incubation period. Additionally, the incorporation of MMT significantly improved the mechanical and thermal properties of the BC membranes. The degradation temperature of the composites was extended, and a decrease in the water holding capacity (WHC) and an improvement in the water release rate (WRR) were noted. Determining a cost-effective medium for the production of BC and the characterization of the produced composites are extremely important for the biomedical applications of BC, such as in wound dressing materials.