Multicellular aggregates contain chemical gradients that drive physiological differentiation, contributing to drug tolerance in tumors and infectious biofilms. We study how bacteria survive in multicellular structures, focusing on the pathogen Pseudomonas aeruginosa. Our work examines specialized metabolic pathways and mechanisms of multicellular organization that promote access to resources. Microelectrode profiling and fluorescent reporters show that chemical subzones within P. aeruginosa biofilms correlate with localized metabolic gene expression. Using Stimulated Raman scattering microscopy, we can profile metabolic activity along chemical gradients, and we have uncovered a complex interplay of metabolic strategies. These include the cross-feeding of metabolites, such as endogenous lactate and phenazines. We have also shown that, in addition to promoting redox homeostasis, phenazine metabolism, in particular, enhances antibiotic tolerance in biofilm cells. Furthermore, localization within the biofilm also affects the production of a newly discovered virulence factor called the R-body, a large extendable polymer that is toxic to eukaryotic hosts. Together, these observations reveal the diverse ways in which multicellular physiology and behavior can enhance the robustness and pathogenicity of biofilm-based P. aeruginosa infections. Techniques that reveal the chemical and spatial variation within multicellular structures across depth provide insights that are specific to the communal lifestyle, and these insights have the potential to inform efforts to treat recalcitrant diseases.