Two new 4'-acetylated analogs of chrysomycin were discovered during the screening for antitumor agents from the metabolites of actinomycetes. Their structures and physicochemical properties were determined by standard spectrometric analyses. Their cytotoxicities and antimicrobial activities were evaluated against a panel of cancer cell lines and microbes. While acetylation reinforced the cytotoxicity of chrysomycin B, it weakened the activity of chrysomycin A. Chrysomycin A and its acetylated analog showed high cytotoxicity toward most of the cancer cells with IC50s less than 10 ng ml-1. The 4'-acetyl-chrysomycin A was predominantly observed in nuclei at concentrations where the autofluorescence was observable. Chrysomycins were effective toward Gram-positive bacteria. The 4'-acetylated-chrysomycin A and B had MICs of 0.5-2 μg ml-1 and 2 to greater than 64 μg ml-1, respectively, toward Gram-positive bacteria including MRSA and VRE.