Background: Bacterial cellulose (BC) is produced by certain acetic acid bacteria (AAB) and has gained increasing interest due to its versatility and possible applications in various industrial sectors including biomedical, cosmetic and food packaging. However, the high production cost and low BC yield remain an issue. Therefore, it is necessary to develop novel strategies for effective and inexpensive BC production. Methods: The aim of this study was to assess the capability of five different strains of AAB belonging to the species Komagataeibacter xylinus, Komagataeibacter rhaeticus, and Komagataeibacter hansenii to grow and produce BC on various agri-food by-products: beet molasses, brewery spent grains and cheese whey. The assessment of growth and BC production by the selected strains was done during the incubation of the samples at 28 °C for 10 days. Results: The results indicated that all the AAB strains were able to grow on all the natural substrates although different growth kinetics was observed. The ability to produce BC was dependent on the AAB strain and the agrifood by-product. Brewery spent grains showed good BC yields by all the strains considered. Conversely, on beet molasses, the highest BC yields were obtained with the strain K. xylinus DSM 2004, reaching 3 g/L of dried pellicle. Whey and molasses were the most selective substrates regarding BC yields; indeed, only the strains K. xylinus DSM 2004, K. rhaeticus LM4, and K. rhaeticus SM1 were able to produce significant amounts of BC. Generally, the BC yield gradually increased with the increase of incubation time, but the rate of BC yield remained almost constant after 7 days in all the substrates considered. Conclusions: This work allowed to identify the strains capable of producing the highest quantities of bacterial cellulose in relation to the agri-food by-product used as substrate; however, certain growth variables could be modulated in order to potentially increase and optimize the yields of bacterial cellulose.