One hundred and twenty-six mesophilic Actinomycete cultures were isolated from the Aegean region of Turkey. The antimicrobial activities of pure isolates were tested using the agar-plaque method. Based on high antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli O157-H7 (E. coli), the isolate M-33-5 was selected for bioactivity-guided isolation. Fermentation, followed by solvent partition (H2O-EtOAc, H2O-n-BuOH), showed that the highest activity was present in the EtOAc extract. By using chromatographic methods, two bioactive compounds were isolated and their structures were determined by spectral methods to be 4'-deacetyl griseusin A and griseusin A. The MIC values of griseusin A and 4'-deacetyl griseusin A against MRSA and E. coli were < or = 1.0 microg/mL. The cytotoxicities of the EtOAc extract and 4'-deacetyl griseusin A were also evaluated against two cancer cell lines (human servical cancer: HeLa; murine fibroblastic cells: L-929). The EtOAc extract showed strong cytotoxic activity against HeLa and L-929 lines with IC50 values of 1.57 and 2.43 microg/mL, respectively, whereas 4'-deacetyl griseusin A was very potent with IC50 values of 0.43 versus HeLa, and 0.12 microg/mL against L-929. The active strain M-33-5 was identified as Streptomyces griseus by 16SrDNA sequence data.