Two new piperazine-2,5-dione derivatives, gliocladride A (1) and B (2), along with deoxymycelianamide (3) were isolated from the mycelial mass of marine fungus Gliocladium sp. Their structures were established on the basis of spectral data, and the stereochemical assignments were made by chiral HPLC analysis of the hydrolyzed compounds and optical rotation comparison with known compound. Their cytotoxic activity was tested on three cancer cell lines, HL-60, U937 and T47D by MTT assay. As a result, gliocladride A (1) and B (2) showed moderate cytotoxic activity against the three cell lines with IC50 values from 11.60 mg/ml to 52.83 mg/ml, while deoxymycelianamide (3) showed strong cytotoxic activity against U937 cell line with IC50 values of 0.785 mg/ml.