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Much of our understanding of bacterial behavior stems from studies in liquid culture. In nature, however, bacteria frequently live in densely packed spatially-structured communities. How does spatial structure affect bacterial cooperative behaviors? In this work, we examine rhamnolipid production—a cooperative and virulent behavior ofPseudomonas aeruginosa. Here we show that, in striking contrast to well-mixed liquid culture, rhamnolipid gene expression in spatially-structured colonies is strongly associated with colony specific growth rate, and is impacted by perturbation with diffusible quorum signals. To interpret these findings, we construct a data-driven statistical inference model which captures a length-scale of bacterial interaction that develops over time. Finally, we find that perturbation ofP. aeruginosaswarms with quorum signals preserves the cooperating genotype in competition, rather than creating opportunities for cheaters. Overall, our data demonstrate that the complex response to spatial localization is key to preserving bacterial cooperative behaviors.