In this work, the effects of carbon sources and culture media on the production and structural propertiesof bacterial cellulose (BC) have been studied. BC nanofibers were synthesized using Gluconacetobacterxylinus strain PTCC 1734. Media used were Hestrin–Schramm (H), Yamanaka (Y), and Zhou (Z). Fivedifferent carbon sources, namely date syrup, glucose, mannitol, sucrose, and food-grade sucrose wereused in these media. All the produced BC pellicles were characterized in terms of dry weight production,biomass yield, thermal stability, crystallinity and morphology by thermogravimetric analysis (TGA), x-ray diffraction (XRD), and field emission scanning electron microscopy (FE-SEM). The obtained resultsshowed that mannitol lead to the highest yield, followed by sucrose. The highest production efficiency ofmannitol might be due to the nitrogen source, which plays an important role. The maximum improvementon the thermal stability of the composites was achieved when mannitol was used in H medium. Inaddition, the crystallinity was higher in BC formed in H medium compared to other media. FE-SEMmicrographs illustrated that the BC pellicles, synthesized in the culture media H and Z, were stable, unlikethose in medium Y that were unstable. The micrographs of BC produced in media containing mannitol andsucrose provided evidence of the strong interfacial adhesion between the BC fibers without noticeableaggregates.