Bactofilins are a widely conserved protein family implicated in cell shape maintenance and in bacterial motility. We show that the bactofilins BacE and BacF from Bacillus subtilis are essential for motility. The proteins are required for the establishment of flagellar hook- and filament structures, but apparently not for the formation of basal bodies. Functional YFP fusions to BacE and to BacF localize as discrete assemblies at the B. subtilis cell membrane, and have a diameter of 60 to 70 nm. BacF assemblies are relatively static, and partially colocalize with flagellar basal bodies, while BacE assemblies are fewer per cell than those of BacF and are highly mobile. Tracking of BacE foci showed that the assemblies arrest at a single point for a few hundred milliseconds, showing that a putative interaction with flagellar structures would be transient and fast. When overexpressed or expressed in a heterologous cell system, bactofilins can form filamentous structures, and also form multimers as purified proteins. Our data reveal a propensity for bactofilins to form filaments, however, in B. subtilis cells, bactofilins assemble into defined size assemblies that show a dynamic localization pattern and play a role in flagellar assembly.