Pseudomonas aeruginosa infection is a significant threat for clinicians. Increasing incidents of resistant biofilm infection result in high mortality rates worldwide. There is a considerable current interest in the field of extracellular DNA (eDNA)-mediated P. aeruginosa biofilm formation. eDNA acts as a glue to make biofilm more stable. This review focuses on the diverse mechanisms and factors, which enhance the eDNA release into the extracellular milieu. Furthermore, eDNA-mediated molecular interactions within the biofilm are emphasized. In addition, drug resistance mechanisms due to the versatility of eDNA are discussed. Spatial physiological diversity is expected due to different metabolic activity of bacterial subpopulation present in P. aeruginosa biofilm layers. In P. aeruginosa, eDNA release is accomplished by cell lysis and OMVs (outer membrane vesicles). eDNA release is a spontaneous and multifactorial process, which may be accomplished by PQS, pyocyanin, and lambda prophage induction. Hydrogen peroxide and pyocin trigger cell death, which may facilitate eDNA release. Lung mucosa of cystic fibrosis patients is enriched with eDNA, which acidifies biofilm and develops P. aeruginosa resistance to aminoglycosides. Further studies on spatial and molecular characterization of bacterial subpopulation in biofilm will shed light on eDNA-biofilm interaction more precisely.