AbstractPathogenic bacteria form biofilms on epithelial cells, and most bacterial biofilms show increased production of membrane vesicles (MVs), also known as outer membrane vesicles in Gram‐negative bacteria. Numerous studies have investigated the MVs released under planktonic conditions; however, the impact of MVs released from biofilms on immune responses remains unclear. This study aimed to investigate the characteristics and immunomodulatory activity of MVs obtained from both planktonic and biofilm cultures of Pseudomonas aeruginosa PAO1. The innate immune responses of macrophages to planktonic‐derived MVs (p‐MVs) and biofilm‐derived MVs (b‐MVs) were investigated by measuring the mRNA expression of proinflammatory cytokines. Our results showed that b‐MVs induced a higher expression of inflammatory cytokines, including Il1b, Il6, and Il12p40, than p‐MVs. The mRNA expression levels of Toll‐like receptor 4 (Tlr4) differed between the two types of MVs, but not Tlr2. Polymyxin B significantly neutralized b‐MV‐mediated cytokine induction, suggesting that lipopolysaccharide of native b‐MVs is the origin of the immune response. In addition, heat‐treated or homogenized b‐MVs induced the mRNA expression of cytokines, including Tnfa, Il1b, Il6, and Il12p40. Heat treatment of MVs led to increased expression of Tlr2 but not Tlr4, suggesting that TLR2 ligands play a role in detecting the pathogen‐associated molecular patterns in lysed MVs. Taken together, our data indicate that potent immunomodulatory MVs are produced in P. aeruginosa biofilms and that this behavior could be a strategy for the bacteria to infect host cells. Furthermore, our findings would contribute to developing novel vaccines using MVs.