A methylotrophic actinomycetes strain MO-16, which produce the antimicrobial compound, was isolated from soil and supposed as Amycolatopsis sp. based on taxonomic studies. The cell-free extract of methanol-grown strain MO-16 showed dehydrogenase activity for methanol and formaldehyde when various electron acceptors were added for oxidation. On the other hand, methanol did not affect the production of antimicrobial compounds, and organic nitrogen sources such as corn steep liquor and peptone were better than inorganic nitrogen sources. These compounds showed broad antimicrobial spectrum to the tested strains such as bacteria and yeast. The antimicrobial comounds were very stable under heat(121$^{circ}C$), acid(pH2.0), alkali(pH11.0) treatments. These compounds were isolated by ethylacetate extract, silica gel column chromatography and reverse phase HPLC. Two compounds(peak 1 and 2) were detected as antimicrobial compounds through the HPLC analysis. The peak 2 was purified as a single compound and revealed a 98% purity.