The antifungal activity of Ailanthus excelsa was evaluated using human pathogenic organism (Aspargillus niger, Aspargillus flavus, Aspargillus fumigatus, and penicillium notatum). The Plant reported to contain chemical constituents like quassinoids excelsin, glaucarubin, ailanthone, glaucarubinone, glaucarubolone. This study involves the preliminary screening, quantitative determination and the qualitative thin layer chromatographic separation of secondary, metabolites from different part of plant. The chloroform fraction of methanol extract exhibited significant broad spectrum of inhibition at 500mg/ml concentration on fungi.