The rising consciousness about the benefits of environment friendly and biodegradable materials demand a substitute for the prevailing non-degradable and toxic materials. Towards this aspect bacterial cellulose (BC) has numerous potential applications owing to their vital properties like high biocompatibility, biodegradability, and ecofriendly nature. In the present study, we have synthesized BC using Komagataeibacter sucrofermentans strain through batch fermentation and functionalized it with sulfate groups to form bacterial cellulose sulfate (BCS) that has negligible reports so far. Using this BCS, we have synthesized a highly transparent film by drop casting method, which exhibited high optical transmittance of 90–92% in the visible wavelength range. BCS was thoroughly characterized using SEM and AFM for its surface morphology. The XPS, FTIR, TGA and XRD studies were performed to confirm the successful sulfonation, stability and crystallinity of BC and BCS. The film morphology, surface roughness and mechanical properties were monitored using SEM, AFM, and Instron universal testing machine. The results confirm smooth surface with good integrity and mechanical properties of the BCS film. Furthermore, cell viability studies confirmed the biocompatible nature of the sample. Owing to these salient features, BCS films hold tremendous potential in the field of biomedicine, optoelectronics as well as food packaging.