Abstract The significance of extracellular DNA (eDNA) in biofilms was overlooked until researchers added DNAse to a Pseudomonas aeruginosa biofilm and watched the biofilm disappear. Now, a decade later, the widespread importance of eDNA in biofilm formation is undisputed, but detailed knowledge about how it promotes biofilm formation and conveys antimicrobial resistance is only just starting to emerge. In this review, we discuss how eDNA is produced, how it aids bacterial adhesion, secures the structural stability of biofilms and contributes to antimicrobial resistance. The appearance of eDNA in biofilms is no accident: It is produced by active secretion or controlled cell lysis - sometimes linked to competence development. eDNA adsorbs to and extends from the cell surface, promoting adhesion to abiotic surfaces through acid-base interactions. In the biofilm, is it less clear how eDNA interacts with cells and matrix components. A few eDNA-binding biomolecules have been identified, revealing new concepts in biofilm formation. Being anionic, eDNA chelates cations and restricts diffusion of cationic antimicrobials. Furthermore, chelation of Mg(2+) triggers a genetic response that further increases resistance. The multifaceted role of eDNA makes it an attractive target to sensitize biofilms to conventional antimicrobial treatment or development of new strategies to combat biofilms.