A new asymmetric diketopiperazine dimer, Asperflocin (1), has been isolated from the fungus Aspergillus versicolor 16F-11. It's structure was determined by virtue of detailed spectroscopic analysis, Marfey's method, and 13C NMR chemical shifts, [α] 25 D, and TDDFT/ECD calculations. Cytotoxic activity test showed that 1 has moderate cytotoxic activity against A375 cell line with IC50 value of 10.29 ± 2.37 μM.