Pseudomonas aeruginosa is an opportunistic human pathogen, capable of colonizing a wide range of tissue causing acute and chronic infections. The microorganism significantly contributes to morbidity and mortality in respiratory infections among immunocompromised patients with cystic fibrosis. Besides, the gram-negative pathogen is a common cause of nosocomial infections, attributed to the secretion of an extensive array of virulence factors contributing to a successful infection strategy. Bacteria have evolved mechanisms for the secretion of such virulence factors, typically enabling bacterial colonization via direct contact with the host cells. Moreover, P. aeruginosa produces extracellular membrane vesicles (MVs), delivering a wide variety of molecules into the eukaryotic host cell, thereby contributing to its pathogenicity, regardless of being in direct contact. Hence, these membrane-encapsulated structures are considered to represent a novel bacterial secretion pathway (T0SS), not only being involved in infection but also crucial in inter- and intra-species cell communication. Recent studies revealed the existence of several routes underlying MV biogenesis, resulting in MVs with different biochemical cargos, which in turn affect their biological function. Despite their relevance in virulence, the mechanisms governing MV biogenesis and the cellular machinery restructuring the bacterial membrane remain poorly understood. Herein, we report the identification of two protein families as key players in restructuring events of both the inner and outer membrane. Deletion and overexpression of the genes encoding these proteins strongly impacted the vesiculation in P. aeruginosa. MV characterization of the constructed strains was conducted by multiple methods encompassing Nanoparticle Tracking Analysis (NTA), fluorescent membrane staining and electron microscopy analysis. Understanding the processes of vesicle formation and elucidating the protein machinery responsible for membrane remodeling offers valuable insights for developing targeted therapeutics against infections caused by P. aeruginosa.