We have shown previously that extracellular DNA (eDNA) is required for the formation of sessile, hydrated biofilms of Pseudomonas aeruginosa playing a critical role during the early stages of biofilm development1 . Interestingly, the mechanism of eDNA production during these early stages has yet to be elucidated. We have recently determined that interstitial biofilms of P. aeruginosa that form at the interface of a glass coverslip and semi-solid nutrient media contain high levels of eDNA2 . Interstitial biofilms formed under these conditions contain a zone at the leading edge that is comprised of an actively migrating monolayer of cells. We have exploited this feature to explore the mechanism of eDNA production using advanced microscopy techniques. We cultured interstitial biofilms in the presence of a cell impermeant eDNA stain and acquired high resolution phase contrast and fluorescence microscopy images at high frame rates. These time-series revealed that eDNA production is released in bursts that are concomitant with cellular explosions. Interestingly, this process of explosive cell lysis involves the transition of rod-shaped cells into spherical cells that ultimately explode to release their cellular contents including genomic DNA. We used super-resolution microscopy to explore this process further and found that explosive cell lysis also accounts for the production of large numbers of membrane vesicles (MVs) that form through the re-annealing of shattered membranes. This process captures cellular contents present in the immediate vicinity into the MVs. We have also determined that explosive cell lysis contributes to eDNA release during the early stages of the development of hydrated sessile biofilms of P. aeruginosa. In summary, we have found that a novel mechanism of explosive cell lysis releases eDNA and membrane vesicles in P. aeruginosa biofilms and may also account for the presence of moonlighting cytoplasmic proteins on the bacterial cell surface and in the extracellular milieu.