To isolate and track the active substances against citrus blue and green mold from wide Gymnopus sp. （0612-9）. Methods： The phase silica gel column chromatography， ODS column chromatography and semi-preparative HPLC were adopted to separate its active constituents. A developed TLC-bioautography （agar casting） was invented to track these active constituents with verification from the Oxford cup method， TLC-UV fluorescence method， colorimetric method with 10% sulfuric acid-ethanol. Results： Five substances were prepared. There were four antifungal active substances （Ⅰ1.1，Ⅰ2， Ⅰ3 andⅡ） tracked by the way of the developed TLC-bioautography. Detect able amount was even less than 18.6 μg/mL. Conclusion： The method has the advantages of high clarity and accuracy， and is suitable for guiding the separation of moderately polar active substances such as the antifungal active substances from Gymnopus sp.