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The fungus Trichosporum sp. is an endophytic fungus isolated from seeds of Trigonella foenum-graecum (Fabaceae). The endophytic fungus Trichosporum sp. has been subjected to chemical and biological investigations resulted in the isolation of two new diketopiperazine alkaloid isomers identified chemically as;(6-S)-3-(1, 3-dihydroxypropyl)-6-(2-methylpropyl) piperazine-2, 5-dione and(6-R)-3-(1, 3-dihydroxypropyl)-6-(2-methylpropyl) piperazine-2, 5-dione (1 and 2). Three chiral centers in both of 1 and 2 were not identified due to lack of the isolated amounts. The isolated compounds were identified using different 1D and 2D NMR as well as IR and HR-MS techniques. The isolated compounds were examined for in vitro antileishmanial, antifungal, antibacterial and antimalarial activities. Compound 1 and 2 showed antileishmanial activities with IC50 values of 96.3 and 82.5 µg/ml, respectively.