This study investigates the impact of cellular arrangement on metabolic activity and antibiotic tolerance in *Pseudomonas aeruginosa* biofilms. The researchers found that *P. aeruginosa* biofilms form vertical striations packed lengthwise across most of their depth, with clonal cells arranged in a specific pattern. Genetic mutants, particularly those defective in pilus function and O-antigen attachment, show altered cellular packing. The study demonstrates that cellular arrangement affects resource access within the biofilm, leading to uneven distribution of substrates and metabolic activity. Additionally, altered cellular arrangement influences the survival of resident cells and the susceptibility of subpopulations to antibiotics. The findings provide insights into the cellular features that determine biofilm microanatomy and its consequences for physiological differentiation and drug sensitivity.This study investigates the impact of cellular arrangement on metabolic activity and antibiotic tolerance in *Pseudomonas aeruginosa* biofilms. The researchers found that *P. aeruginosa* biofilms form vertical striations packed lengthwise across most of their depth, with clonal cells arranged in a specific pattern. Genetic mutants, particularly those defective in pilus function and O-antigen attachment, show altered cellular packing. The study demonstrates that cellular arrangement affects resource access within the biofilm, leading to uneven distribution of substrates and metabolic activity. Additionally, altered cellular arrangement influences the survival of resident cells and the susceptibility of subpopulations to antibiotics. The findings provide insights into the cellular features that determine biofilm microanatomy and its consequences for physiological differentiation and drug sensitivity.