We aimed to determined that characterization of antimicrobial compounds of Actinomycete isolates (A32, A33) which were determined have antimicrobial activities in a previous study of ours. Different fermentation media were used to determination effective antimicrobial compounds production of AA32 and AA33 by fermentation.Fermentation procedure were performed in three major steps: sporulation, inoculation, and fermentation.Effective antimicrobial molecules of AA32 and AA33 to determined as test microorganisms Staphylococcus aureus ATCC 25923 and Esherichia coli ATCC 25922 were used during fermentation. Raw materials extraction of AA32 and AA33 were performed by solvent extraction and active compound determination were performed by using TLC, biootography, colon chromatography and UV spectrophotometer methods.Also to describe these two isolates, fatty acid profiles were determined using gas chromatography. As conclusion, The isolate AA32 was identified as Streptomyces-halstedii-olivaceus at a similarity index of 0.154 and the isolate AA33 was identified as Streptomyces californicus at a similarity index of 0.540 by the MIDI system. The antimicrobial compound isolated has O-H and/or N-H rich, having amine groups, has an Rf value as 0.527, its λmax is at 254 nm, and a polar compound. Its UV spectrum peak and, its λmax bring to mind the cephalosporin or cephalosporin like aromatic or polyene compounds