Glycogen is mainly found as the principal storage form of glucose in cells. Many bacteria are able to synthesize large amounts of glycogen under unfavorable life conditions. By combining infrared spectroscopy, single molecule force spectroscopy (SMFS) and immuno-staining technique, we evidenced that planktonic P. fluorescens (Pf) cells are also able to produce glycogen as an extracellular polymeric substance. For this purpose, Pf suspensions were examined at 3 and 21 h of growth in nutritive medium (LB, 0.5 g/L). The conformation of the extracellular glycogen, revealed through its infrared spectral signature, has been investigated by SMFS measurements using Freely Jointed Chain model. The analysis of force versus distance curves showed over growth time that the increase of glycogen production was accompanied by an increase in glycogen contour lengths and ramifications. These results demonstrated that the production of extracellular bacterial glycogen can occur even if the cells are not subjected to unfavorable life conditions.