and Purpose: Aspergillus genus biosynthesize a large number of metabolites with wide-ranging bioactivities, but how to target the active compounds quickly and accurately? In this work, we provide a strategy to fast identify bioactive metabolites in fungi with similar morphology but different antimicrobial activities. Materials and Methods: Combined with High Performance Liquid Chromatography (HPLC) profiles and bioactivity screening of fungal extracts, three Aspergillus sp. strains YSN038,

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... 2 and YSN064 were studied. The bacteria Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, Kocuria rhizophila, Escherichia coli and Ralstonia solanacearum were used for antibiotic assay.Results: Three strains showed the similar morphology, but the crude extracts of YSN038 and YSN064 displayed antibacterial activities against B. pumilus, B. subtilis, S. aureus and K. rhizophila, whereas YSN052 had not. The compound contributed to antibacterial activity in vitro was rapidly identified, isolated and characterized. Compound 3 connected with specific HPLC peak should be the antibiotic substance by analysis of HPLC profiles, and was confirmed after the following antimicrobial tests. The compound was elucidated as butyrolactone I based on nuclear magnetic resonance(NMR) and mass spectrometry (MS) data, and showed various biological activities.Conclusions: Our study has significant scope on targeting antibacterial metabolites. The described method could also be used as a rapid and cost-effective tool for screening other bioactivity products. As such, this article could offer a fast approach to isolate drug-lead compounds from microorganisms.