A series of diketopiperazine derivatives (1–12) were evaluated for their in vitro cytotoxic activity against Leishmania donovani promastigotes and amastigotes. Cytotoxicity study revealed that the number and types of the substituents in the phenyl rings have valuable influence on cytotoxic activity. Compounds 1, 3, 4, 11, and 12 demonstrated appreciable cytotoxic activities with the mean IC50 values 1.4, 1.1, 1.02, 1, 0.7 lg/ml on extra cellular promastigotes and 1.6, 1.8, 0.61, 0.53, 1.1 lg/ml on intracellular amastigotes, respectively. The results suggested that diketopiperazine derivatives 4, 11, and 12 could be envisaged as new entrants in the domain of antileishmanial agents.