Strain JX22, exhibiting a broad range of antimicrobial activities to fungal pathogens, was isolated and classified as representing Pseudomonas kilonensis. In this study, the mutant JX22MT1 was obtained by the EZ-Tn5 transposon mutation and showed no antifungal activity against Fusarium oxysporum f. sp. lycopersici as compared with wild-type strain JX22. The pqqC gene was disrupted in the mutant. Antifungal activity at the wild-type level was restored from the mutant JX22MT1 with the introduction of the functional pqqC gene, which encodes pyrroloquinoline–quinone synthesis protein C. The results suggest that pqqC is essential for antifungal activity of P. kilonensis JX22 against F. oxysporum f. sp. lycopersici.