Among the adaptive response of bacteria to rapid changes of environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms had been elucidated with which bacteria change their cell surface and membranes in the presence of different environmental stresses. Within these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has gained particular research interest because of its involvement in pathogenic processes such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long chain alcohols, osmotic stress caused by NaCl, EDTA, and after heat shock cells of this strain released MV within 10 min in the presence of a stressor. The formed MV showed similar size and charge properties as well as comparable composition in proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity and an enhanced tendency to form biofilms was demonstrated with this study. Therefore, the release of MV as stress response could be put in a physiological context.