Two novel cyclic hexapeptides designated actinosynneptides A (1) and B (2), together with three tryptophan containing diketopiperazines, namely cyclo(L-Trp-L-Trp) (3), cyclo(L-Trp-N-MeL-Trp) (4), and cyclo(N-MeL-Trp-N-MeL-Trp) (5), were isolated from the culture of the genetically engineered strain HGF052::asm18 derived from Actinosynnema pretiosum ATCC31565. Their structures were elucidated on the basis of spectroscopic analyses and single-crystal X-ray diffractions. Compound 1 is the first example of 3-amino-6-hydroxy-2-piperidone-containing cyclic peptides, and 1 and 2 showed moderate cytotoxic activities against HeLa and PC3 cell lines.