Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, exists in both biofilm and planktonic lifestyles, with biofilms playing a crucial role in chronic infections. Extracellular vesicles (EVs), secreted in both states, are known to mediate intercellular communication, but their compositional and functional differences remain poorly understood. This study systematically investigated the physicochemical properties and proteomic profiles of EVs derived from both planktonic and biofilm. Proteomic profiling revealed distinct signatures, with biofilm-derived EVs (BEVs) enriched for virulence factors and proteins associated with bacterial adhesion and iron acquisition, including FptA, PA4218, PhzB2, PhzS, and PhzG1. Functional assessments indicated that these compositional differences influence bacterial behaviors, particularly affecting biofilm adhesion, antibiotic tolerance, and iron chelation. The role of EV-associated siderophores was specifically investigated; however, determining whether they function as direct iron ion transporters proved inconclusive. Although these iron carriers induced downregulation of the fur gene—a key regulator of iron homeostasis—this effect occurred independently of external iron availability, suggesting a potential signaling role beyond simple iron transport.