Plant pathogenic Pseudomonas species produce effectors, toxins and cyclic lipopeptides to infect various host plants. Despite many studies aiming to understand the underlying mechanisms of virulence in Pseudomonas spp., the function of genes in the srf gene cluster including srfC , which was formerly known as HopL1, remains undetermined. To investigate the roles of srf genes in the virulence of the bacterial pathogen, each srfA , srfB , srfC and srfD gene from the srf cluster of Pseudomonas cichorii JBC 1 was knocked out. When tomato seedlings were infected with the knockout mutants by flood inoculation, disease incidence was suppressed only in srfC ‐defective mutants (∆ srfC ) compared to wildtype. Interestingly, when the ∆ srfC strain was inoculated directly into the apoplast of tomato leaves by vacuum infiltration, disease developed similar to that of the wildtype. In addition, the ∆ srfC strain showed defective swarming motility and biofilm formation, and the attachment of ∆ srfC cells on the leaf surface was significantly reduced compared to wildtype. Furthermore, droplets of the ∆ srfC culture supernatant did not spread on a hydrophobic surface, unlike the wildtype. The results indicate that the SrfC protein plays important roles in swarming motility, biofilm formation and attachment/colonization on host surfaces; thus, it is beneficial for the pathogen's dispersion and entry into host tissues and consequently contributes to disease development. This study elucidates for the first time the functional role of srfC in P. cichorii virulence. The results will broaden understanding of plant and bacterial pathogen interactions.