The role of fimbrial and afimbrial adhesins of Xylella fastidiosa in biofilm formation was assessed by visualization of cell aggregates of mutant strains after incubation on glass surfaces. FimA- or FimF-fimbrial mutants adhered as solitary cells at a slightly lesser frequency to glass surfaces than the parental strain; however, cell aggregates were not formed, unlike the wild-type strain. Conversely, whereas the XadA- and HxfB- nonfimbrial mutants also exhibited a much lower frequency of adherence to glass surfaces than the wild-type strain, most of the cells retained on the surfaces were in cell aggregates of different sizes, much like that of the parental strain. Neither fimbrial or afimbrial mutants formed a mature biofilm on the sides of flasks of broth cultures, unlike the dense biofilm formed by the wild-type strain. Although FimA- and FimF- mutants did not form cell aggregates on glass surfaces when incubated as individual strains, aggregates of a FimA- or FimF- mutant were observed when co-incubated with either a XadA- mutant or HxfB-mutant, respectively. These results are consistent with a model in which the fimbrial adhesins FimA and FimF are involved preferentially in cell-to-cell aggregate formation whereas the afimbrial adhesions XadA and HxfB preferentially contribute to initial cell binding to surfaces, whereupon further cell aggregation can occur. In each of five separate experiments, FimA, FimF, XadA, and HxfB mutants of X. fastidiosa all were less virulent to grape than the corresponding wild-type strain. Fimbrial and afimbrial mutants might produce a reduced biofilm within vessels of grape and, hence, be deficient in various cell-density-dependent traits required for movement through the plant and, thus, virulence.