Cyclo(L-Pro- L-Asp) diketopiperazine was synthesized and its antimicrobial properties were compared against its 2-ketopiperazine analog. The results indicate the significance of the peptide bond in antimicrobial property of cyclo(L-Pro- L-Asp) against various pathogenic bacteria and fungi with potent inhibitory effect over the 2-ketopiperazine analog. Cyclo(L-Pro- L-Asp) exhibited a higher inhibitory activity against Penicillium expansum (MIC 8 lg/mL) than the standard fungicide amphotericin B (MIC 64 lg/mL). The most potent activity by cyclo(L-Pro-L-Asp) was exhibited against Trichophyton rubrum (MIC 2 lg/mL).