In　this　study,　antibacterial　substances　were　extracted　and　identified　from　the　mycelia　of　Eurotium　cristatum　FS-1,　and　evaluated　for　antibacterial　activity　against　Pseudomonas　putida　LP-3.　The　underlying　mechanism　was　investigated　by　microscopic　observations,　LP-3　cell　content　analysis,　and　metabolomic　analysis.　The　results　showed　that　the　half　maximal　inhibitory　concentration　(IC50)　of　the　ethyl　acetate　extract　(FSEAE)　from　the　mycelia　of　Eurotium　cristatum　FS-1　on　P.　putida　LP-3　(1　×　104　CFU/mL)　was　8　mg/mL.　As　observed　by　scanning　electron　microscopy　(SEM),　FSEAE　destroyed　the　cell　membrane　of　P.　putida　LP-3.　The　K+　content　in　the　culture　supernatant　of　P.　putida,　the　activity　of　oxidative　stress　enzymes　in　the　cell　metabolic　pathway　(superoxide　dismutase,　glutathione　peroxidase,　and　catalase)　and　the　content　of　malondialdehyde　(MDA)　were　measured,　showing　that　the　antibacterial　effect　of　FSEAE　mainly　in　the　logarithmic　growth　period　of　P.　putida　LP-3　(8–16　h).　Non-targeted　metabonomics　was　used　to　analyze　FSEAE　treated　samples　and　control　samples　(treated　by　dimethyl　sulfoxide),　and　a　total　of　124　metabolites　were　obtained,　of　which　30　metabolites　were　significantly　different　between　the　two　groups.　Kyoto　Encyclopedia　of　Genes　and　Genomes　(KEGG)　enrichment　analysis　showed　that　FSEAE　affected　the　growth　and　metabolism　of　P.　putida　mainly　by　interfering　with　the　tricarboxylic　acid　cycle.　The　results　from　this　study　can　provide　a　theoretical　reference　for　the　development　and　utilization　of　Saccharomyces　coronatus　and　its　metabolites.　©　2023　Chinese　Chamber　of　Commerce.　All　rights　reserved.