Xylella fastidiosa is the causal agent of Pierce's disease (PD) in grapevines. It has type I and type IV pili, which are both virulence factors involved in the PD-associated processes of motility, aggregation, and biofilm formation. Many questions remain as to how the two pili are regulated. We previously identified a X. fastidiosa pil-chp chemosensory-like cluster as an operon composed of genes pilG-I-J-L-chpB-C. In this study, we deleted pilG (resulting in a Delta pilG-I strain) and pilJ and discovered that both mutants (Delta pilG-I and pilJ) had reduced virulence after 24 weeks post-inoculation, whereas Delta chpB and Delta chpC did not. Both Delta pilG-I and Delta pilJ lost motility and were impaired in biofilm formation in rich artificial media and xylem sap. Gene expression was significantly downregulated for representative fimbrial adhesin and motility genes in Delta pilG-I, and to a lesser extent in Delta pilJ. Our data suggest that Pil, but not Chp, proteins are virulence factors, and pilG-I-J are involved in transcriptional regulation of type I and IV pili virulence genes and therefore motility and biofilm formation. To our knowledge, this is the first report of a chemotaxis-like operon involved in the regulation of key structural genes of both type I and type IV pili.