Bacterial cellulose is identical in chemical composition to cellulose extracted from lignocellulosic biomass, but with partial difference in structural characteristics. These differences, specifically its purity, make it valuable, but its production processes are quite expensive. In the present work, spent black liquor resulting from cotton pulping, as a major industrial waste stream, was investigated as an alternative carbon source in the production of bacterial cellulose (BC) using Acetobacter xylinum. XRD results of the produced cellulose showed that the crystallinity of the BC was lower than that of cotton pulp alpha-cellulose. SEM evaluation confirmed the nano-size of the produced cellulose, while its structure was evidenced by FT-IR analysis. The effect of altering the culture media on some structural features of the produced BC was thoroughly discussed and it was suggested that the spent liquor could be added in amounts of up to 25% for BC production in standard cultures (HS).