Holliday　junction　(HJ)　is　a　four-way　structured　DNA　intermediate　in　processes　of　homologous　recombination　and　DNA　double-stranded　break　(DSB)　repair.　In　bacteria,　HJs　are　processed　via　either　the　RuvABC　or　RecG-dependent　pathways.　In　addition,　RecG　also　plays　a　critical　role　in　the　reactivation　of　stalled　replication　forks,　making　it　an　attractive　target　for　antibacterial　drug　development.　Here,　we　conducted　a　high-throughput　screening　targeting　the　RecG　helicase　from　a　common　opportunistic　pathogen　Pseudomonas　aeruginosa　(Pa).　From　a　library　containing　7920　compounds,　we　identified　Ebselen　and　TPI-1　(2′,5′-Dichloro-[1,1′-biphenyl]-2,5-dione)　as　two　potent　PaRecG　inhibitors,　with　IC50　values　of　0.31　±　0.02　μM　and　1.16　±　0.06　μM,　respectively.　Further　biochemical　analyses　suggested　that　both　Ebselen　and　TPI-1　inhibited　the　ATPase　activity　of　PaRecG,　and　hindered　its　binding　to　HJ　DNA　with　high　selectivity.　These　compounds,　when　combined　with　our　previously　reported　RuvAB　inhibitors,　resulted　in　more　severe　DNA　repair　defects　than　the　individual　treatment,　and　potently　enhanced　the　susceptibility　of　P.　aeruginosa　to　the　DNA　damage　agents.　This　work　reports　novel　small　molecule　inhibitors　of　RecG,　offering　valuable　chemical　tools　for　advancing　our　understanding　of　RecG＇s　function　and　mechanism.　Additionally,　these　inhibitors　might　be　further　developed　as　promising　antibacterial　agents　in　the　fight　against　P.　aeruginosa　infections.　©　2024　Elsevier　Inc.