[Objective] Ralstonia solanacearum is the causative agent of a devastating bacterial wilt disease in solanaceous plants. The purpose of this work was to identify genes involved in the pathogenesis of R. solanacearum. [Methods] We used a Tn5-based mutagenesis strategy to generate random insertion mutants that were then assayed for biofilm formation, cell motility and pathogenicity. Thermal asymmetric interlaced PCR (TAIL-PCR) was performed to identify the Tn5 insertion site of mutants with altered phenotypes. [Results] A total of 400 mutants were generated in the model strain GMI1000. Two mutants were found to be unable to form biofilms and had reduced swimming and swarming motility on soft agar media. When inoculated into tomato plants, both mutants failed to cause wilting disease symptoms. Both mutants carried a Tn5 insertion within the NADH dehydrogenase subunit F gene (nuoF), and the insertion sites were at 103 bp and 225 bp from the translational start site. A complemented strain expressing nuoF under the control of the ripAY promoter fully restored the wild-type phenotype. [Conclusion] The NADH dehydrogenase complex is the first enzyme in the respiratory electron transport chain in microbes. Our data here indicated that NADH dehydrogenase plays a role in biofilm formation, cell motility and pathogenicity in R. solanacearum.