Like many bacteria, Bacillus subtilis possesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: an ftsA deletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzyme in vivo. Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slowand fast-moving molecules in live cells.