Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Deltaorf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Deltaorf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Deltaorf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Deltaorf3 mutant. The phenotypes of the Deltaorf3 mutant and an AHL synthesis (DeltapsyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Deltaorf3 mutant. The swarming motility of the Deltaorf3 mutant was greater than that of the DeltapsyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Deltaorf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605.