Xylella fastidiosa (Xf) produces extracellular DNA in PD3 liquid medium. This extracellular DNA could enhance biofilm formation, a factor in successful establishment of Xf in planta. The relative amounts of extracellular DNA were positively correlated with planktonic growth and biofilm formation in vitro, but were negatively correlated with cell viability. DNase I treatment of actively growing Xf cultures in PD3 medium decreased or inhibited biofilm formation. In contrast, addition of Xf genomic DNA promoted biofilm formation. These results suggest that biogenesis of extracellular DNA may play a role for Xf biofilm formation and could be a critical step in establishment of host-bacterium interaction.