Klebsiella pneumoniae is a leading cause of nosocomial infections, manifesting as a variety of diseases including pneumonia, urinary tract infections and septicemia with a high mortality rate particularly in immune-compromised patients. The formation of biofilms is a key virulence mechanism in K. pneumoniae providing the bacterium with increased resistance to antimicrobial treatments. Our analysis of K. pneumoniae strains revealed large differences in biofilm formation that were independent of mrk operon carriage (mrk encodes type 3 fimbriae) and capsule type. We hypothesized that extracellular DNA (eDNA) forms a major component of biofilm structure. To determine whether removing eDNA affects biofilm formation by K. pneumoniae AJ094, a strain with an enhanced capacity to form biofilms, was studied. Biofilm formation was quantified using a microtiter plate biofilm assay with crystal violet staining. Bacteria were treated with various concentrations of DNase either at the time of inoculation for a period of 8 hours, or to established biofilms after 24 hours of incubation. Biofilms containing bacterial cells and eDNA were visualized by confocal microscopy using the fluorescent dyes SYTO-60 and TOTO-1. DNase treatment (as low as 2.5 µg/mL) reduced early-phase biofilm formation by more than 50%, demonstrating the importance of eDNA in initial biofilm stability. Conversely, established biofilms were not disrupted following DNase treatment. The excretion of eDNA by bacteria residing in biofilms was demonstrated using confocal microscopy. In conclusion, eDNA secretion or release represents a target to prevent K. pneumoniae biofilm formation.