Xylella fastidiosa colonizes the xylem of various host plants, causing economically important diseases such as Pierce's disease in grapevine and citrus variegated chlorosis (CVC) in sweet oranges. The aggregative nature of this bacterium has been extensively documented in the plant xylem and the insect's foregut. Structured communities of microbial aggregates enclosed in a self-produced polymeric matrix and attached to a surface are defined as biofilms. In this study, we characterized biofilm formation by X. fastidiosa through the use of a novel in vitro assay for studying biofilm growth in a potential mimic system of what might occur in planta. We used wood, a xylem rich material, as a surface for bacterial attachment and biofilm formation, under shear force. We demonstrated that X. fastidiosa strains isolated from various hosts formed biofilm on wood in this in vitro assay. Different biofilm morphology was detected, which seems to vary according to the strain tested and microenvironmental conditions analyzed. We observed that strains from different hosts could be grouped according to three parameters: biofilm morphology, the ability to form clumps in liquid culture, and the ability to attach to glass surfaces. We hypothesize that biofilm formation is likely a major virulence factor in diseases related to X. fastidiosa, bringing a new perspective for disease treatment.