Bacterial cellulose (BC) is a highly pure biomaterial that can be produced from agro-industrial residues, making it a sustainable candidate for tissue engineering applications such as biocompatible hydrogel. However, pure BC is rigid and brittle, which limits its clinical handling. This study aimed to synthesise BC membranes from the non-photosynthetic bacterium Komagataeibacter nataicola (TISTR 975) using mature coconut water as the primary fermentation medium, and to improve its mechanical properties by forming an in situ composite hydrogel with polyhydroxyalkanoate (PHA). Sucrose concentration and cultivation period were systematically varied and analysed using response surface methodology (RSM), which identified the optimal condition as a 7-day cultivation at 50 g/L sucrose, yielding a highly uniform membrane with the best structural integrity. In situ fabrication of the BC/PHA composite, achieved by dispersing PHA powder in the culture medium during synthesis, markedly enhanced the material’s flexibility and water-holding capacity. The elongation at break increased from 22.9% for pure BC to 28.2% for the composite, with only a slight reduction in ultimate tensile strength. A cradle-to-gate life cycle assessment (LCA) showed that the BC/PHA membrane had a global warming potential approximately 3.2 times lower than that of bovine collagen membranes, while the incorporation of PHA introduced a negligible additional environmental burden. These findings indicate that the locally producible and biodegradable BC/PHA composite hydrogel offers a favourable balance between mechanical performance, water-holding capacity, and environmental sustainability, positioning it as a promising candidate for the future development of guided tissue regeneration (GTR) gel membranes in dentistry.