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Actinomycin Z
6
(1), a new member of the actinomycin family, along with three congeners of the Z-type (Z
1
, Z
3
, Z
5
) actinomycins, are produced from Streptomyces sp. KIB-H714. Their structures were authenticated by comprehensive spectroscopic data interpretation. Different from all the reported Z-type actinomycins, the β-ring of the new compound actinomycin Z
6
includes an additional ring linked between the actinoyl chromophore and β-peptidolactone. In Z
3
and Z
5
, the L-threonine in β-depsipeptide is replaced by the unusual 4-chlorothreonine, an amino acid rarely found in actinomycin family. All isolates were evaluated for cytotoxicity against five human tumor cell lines and for inhibitory activity against Candida albicans and Staphylococcus aureus.