In this study the effects of growth times and culture mediums on the structural characteristics of bacterial cellulose have been investigated. Bacterial cellulose (BC) nanofibers were synthesized by Gluconacetobacter xylinus. BC pellicles were compared using SEM, FT-IR and X-ray diffraction techniques. The crystallinity index (CrI) and crystallite size (CrS) were calculated based on X-ray measurements. Three growth times (7, 14 and 21 days) and three culture mediums (A, B and C) were applied. SEM micrographs showed that increasing growth time up to 7 days improves the microfibril branches crossing to each other and the number of bundles. However, further increase in growth time (21 days) results in decrease in the microfibril network. On the other hand the hydrogen and C–H bonds were developed by the increase in growth time. In conclusion, BC synthesized in medium B for 7 days had superior properties in terms of CrI, CrS and microfibril networks.