Biofilm is an accumulation of bacterial cells encapsulated in a structurally assembled extracellular polymer matrix that adheres to surfaces. The extracellular polymer matrix is composed mainly of polysaccharides, proteins, and amphiphilic polymers, which are secreted by microorganisms into their environment. Extracellular DNA (eDNA) was recently identified as one of the major components of extracellular polymeric substances (EPSs) and plays an important role in the development of biofilm, especially the early stages, by facilitating initial bacterial attachment and maintaining biofilm structure. More research is needed to fully elucidate the role of eDNA in biofilm development, and such investigations require that extraction of eDNA from the complex biofilm matrix is achieved without any genomic DNA contamination. However, the physical and chemical association of eDNA with extracellular proteins, polysaccharides, and other polymers in the complex matrix of EPS may hinder the liberation of eDNA. Therefore, it is difficult to release eDNA and other materials from the biofilm matrix solely by vortexing or homogenizing, and degradation of certain components of EPSs in the biofilm matrix is necessary in order to release eDNAs that may bind to these compounds. In this protocol, N-glycanase (glycoprotein-degradation hydrolase), dispersin B (biofilm-dispersing glycoside hydrolase), and proteinase K (protein hydrolase) are used to break down biofilm matrix for more efficient and complete extraction of eDNA.