X. fastidiosa, a Gram-negative bacterium that causes disease in many economically important crops worldwide, is naturally competent. This feature allows it to take up DNA fragments from the environment and incorporate them into its own genome, which may lead to host shift and emergence of novel plant diseases. Type IV pili (TFP) play a central role in both natural competence and twitching motility of this bacterium. Many proteins, encoded mainly by pil genes, are responsible for TFP biogenesis and function. However, only few pil genes were evaluated for their role on twitching motility and natural competence in X. fastidiosa. Thus, in this study we aim to assess the role of different pil genes on movement and natural competence of X. fastidiosa by performing site-directed mutagenesis. Results showed that mutation of pilB, pilP, pilR and pilT resulted in loss of twitching motility. In addition, ?pilP and ?pilT mutants lost the natural competence ability, while ?pilB and ?pilR recombined DNA via natural competence at a lower rate than the WT. Overall, results showed that all analyzed genes are essential for twitching motility of X. fastidiosa, while only pilP and pilT are essential for the natural competence feature of this bacterium. Understanding the mechanisms by which this pathogen acquires exogenous DNA and lead to host shift may contribute to the development of new management approaches, mediating the transfer of knowledge to agricultural practices.