The　Holliday　junction　(HJ)　branch　migrator　RuvAB　complex　plays　a　fundamental　role　during　homologous　recombination　and　DNA　damage　repair,　and　therefore,　is　an　attractive　target　for　the　treatment　of　bacterial　pathogens.　Pseudomonas　aeruginosa　(P.　aeruginosa,　Pa)　is　one　of　the　most　common　clinical　opportunistic　bacterial　pathogens,　which　can　cause　a　series　of　life-threatening　acute　or　chronic　infections.　Here,　we　performed　a　high　throughput　small-molecule　screening　targeting　PaRuvAB　using　the　FRET-based　HJ　branch　migration　assay.　We　identified　that　corilagin,　bardoxolone　methyl　(BM)　and　10-(6ˊ-plastoquinonyl)　decyltriphenylphosphonium　(SKQ1)　could　efficiently　inhibit　the　branch　migration　activity　of　PaRuvAB,　with　IC50　values　of　0.40　±　0.04　μM,　0.38　±　0.05　μM　and　4.64　±　0.27　μM,　respectively.　Further　biochemical　and　molecular　docking　analyses　demonstrated　that　corilagin　directly　bound　to　PaRuvB　at　the　ATPase　domain,　and　thus　prevented　ATP　hydrolysis.　In　contrast,　BM　and　SKQ1　acted　through　blocking　the　interactions　between　PaRuvA　and　HJ　DNA.　Finally,　these　compounds　were　shown　to　increase　the　susceptibility　of　P.　aeruginosa　to　UV-C　irradiation.　Our　work,　for　the　first　time,　reports　the　small-molecule　inhibitors　of　RuvA　and　RuvB　from　any　species,　providing　valuable　chemical　tools　to　dissect　the　functional　role　of　each　individual　protein　in　vivo.　©　2022　Elsevier　Ltd