A key problem in understanding major transitions in evolution is the evolution of cooperation: how are mutants that exploit the benefits of cooperation without paying the costs (cheats) suppressed within populations? Biofilms, which display properties of both single cell and multicellular organisms, provide an excellent model system to address this question. Biofilms exhibit grouped population structure – they exist primarily as dense aggregates of cells called microcolonies. We aim to test the hypothesis that cell-grouping displayed by microcolonies in bacterial biofilms provides a mechanism to suppress cheats within the biofilm population. We are using the co-operative trait of siderophore production (an extracellular iron-chelating molecule) within Pseudomonas aeruginosa biofilms to investigate cooperation in biofilms. Under iron-limited conditions, production of siderophores enhanced wild type growth, but microcolonies containing GFP-tagged, pyoverdin-mutant ‘cheats’ developed poorly. In iron-rich conditions, cheats are favoured as siderophore production is costly. With mixed strain biofilms, cheats are dependant on the wild type for successful growth. We suggest that, if cheats reduce colony size, thus self-limiting their global population, microcolony-based group structures may be an important (potentially evolved) mechanism to suppress cheats in bacterial populations