Contrary to multicellular organisms that display segmentation during development, communities of unicellular organisms are believed to be devoid of such sophisticated patterning. Unexpectedly, we find that the gene expression underlying the nitrogen stress response of a developing Bacillus subtilis biofilm becomes organized into a ring-like pattern. Mathematical modeling and genetic probing of the underlying circuit indicate that this patterning is generated by a clock and wavefront mechanism, similar to that driving vertebrate somitogenesis. We experimentally validated this hypothesis by showing that predicted nutrient conditions can even lead to multiple concentric rings, resembling segments. We additionally confirmed that this patterning mechanism is driven by cell-autonomous oscillations. Importantly, we show that the clock and wavefront process also spatially patterns sporulation within the biofilm. Together, these findings reveal a biofilm segmentation clock that organizes cellular differentiation in space and time, thereby challenging the paradigm that such patterning mechanisms are exclusive to plant and animal development. Display omitted •A bacterial segmentation clock patterns biofilm development•Biofilms organize their nitrogen stress response into concentric rings•The concentric ring pattern segments sporulation in space and time•Bacteria use a clock and wavefront mechanism thought exclusive to vertebrates Bacterial biofilms exhibit a clock and wavefront process that spatially patterns sporulation without requiring long-range diffusion of molecular signals and is reminiscent of patterning mechanisms previously thought to be exclusive to plants and animals.