Abstract：The secondary metabolites of Streptomyces ahygroscopicus S91 (CGMCC4.7082) contain various antifungal components. The components that have been identified include tetramycin, nystatin, toyocamycin, albonursin and anisomycin. Through bioinformatics analysis, S. ahygroscopicus S91 genome contains the biosynthetic gene clusters of yunnanmycin and gougerotin. However, these two antibiotics have not been detected in the fermentation products of this strain. During the fermentation culture of Streptomyces ahygroscopicus ΔttmS1, ΔnysB, ΔtymH, a tetracycline, nystatin, and toyocamycin biosynthesis disruption strain, a water-soluble active ingredient inhibiting Aspergillus niger was found in the fermentation broth. The target compound was isolated, guided by antifungal activity. After the fermentation broth was centrifuged and acidified with oxalic acid, the supernatant was decolorized by the macroporous resin DM-2. The decolorization solution was loaded on the 732 resin for cation exchange. Neutral alumina column chromatography and sephadex LH-20 were used for further purification. After the chromatographic treatment, two antifungal compounds A and B were obtained. The compounds were identified by UV, mass spectrometry, and NMR. Compound A was yunnanmycin, and B was either gougerotin or ningnanmycin. The combination of bioinformatics analysis and purification techniques further confirmed the strain’s ability to produce antibiotics such as yunnanmycin and gougerotin.