This protocol provides an experimental model system to study bacterial competition in quasi-spherical three-dimensional bacterial colonies. To do so we encapsulate populations of two fluorescent Escherichia coli strains in inoculation droplets. The inoculation droplets grow when embedded in a hydrogel with nutrients and develop 3D colonies with well-defined sectors-like regions. The protocol can be extended and used with a wide range of different bacteria or – potentially – other cell types.