Sphaerotilus natans is a neutrophilic iron-related sheath-forming filamentous microorganism that presents dual morphotype: single cells and ensheathed cells forming filaments. As S. natans has been proposed as a sorbent for inorganic pollutants and it is occasionally involved in bulking episodes, elucidating factors affecting its filamentous growth is of crucial interest. The purpose of this work was to evaluate the effect of dissolved oxygen (DO) as a factor affecting S. natans filamentation from single cells. A method to quantify S. natans in its filamentous and single-cell morphotypes, based on a differential filtration procedure coupled with quantitative real-time PCR, was developed here. Scanning Electron Microscopy was used to validate the filtration step. Under actively aerated conditions (DO maintained at 7.6 ± 0.1 mg l−1), S. natans grew mainly as single cells throughout the experiment, while a depletion in DO concentration (to ~3 mg l−1) induced its filamentous growth. Indeed, when oxygen was reduced the proportion of single cells diminished from 83.3 ± 5.9  to 14.3 ± 3.4 % while the filaments increased from 16.7 ± 5.9  to 85.7 ± 3.4 %. Our results suggest that oxygen plays a key role in S. natans filamentation and contribute to better understanding of the filamentous proliferation of this bacterium. In addition, the proposed method will be helpful to evaluate other factors favouring filamentous growth.