On the month of March to May 2018, the antimicrobial activity of MxGP 53TM, the isolate of Myxobacteria and SHP 1-2, the Actinobacteria isolate was tested on ten types of microorganisms, consisting of three types of gram-negative bacteria (Escherichia coli, Escherichia coli TolC, and Pseudomonas aeruginosa), four types of gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Mycobacterium smegmatis, and Micrococcus luteus), two types of yeast (Candida albicans and Pichia anomala), and Mucor hiemalis mold. For this purpose, both of these isolates were cultured in 100 mL of liquid medium and incubated in 30^oC with 160-180 rpm followed with the extraction process. The MxGP 53TM was fermented in casitone-yeast extract/HEPES (^CY/_H) culture medium for 10 days and followed by the extraction process using acetone. Meanwhile, SHP 1-2 was fermented in 5294 culture medium for 7 days then extracted with ethyl acetate. The myxobacterial culture extract only works against M. luteus. Meanwhile, the Actinobacteria isolate has antimicrobial activity against all types of gram-positive test bacteria and Mucor hiemalis. Considering that result, the subsequent study was only conducted on the fraction of the Actinobacteria extract against three types of gram-positive bacteria: S. aureus; M. smegmatis; and M. Luteus. For this purpose, the extract was dissolved in methanol followed with the fractionation process using HPLC with the stationary phase of C18 column and the combination of acetone-water as a mobile phase. The fractions were collected in 96-well plate with a retention time of 40 minutes. For the test result, there were three exactly same compounds that were detected in both of S. aureus and M. Smegmatis antimicrobial testing. The LC/MS analysis was showed one compound that has 437,277 774 ^g/_mol molecular mass identified as neoline (C₂₄H₃₉NO₆). Meanwhile, two other compounds which are also have a positive result against both of the test microbes, have a molecular mass of 228.136 16 ^g/_mol (C₁₂H₂₀O₄) and 337.334 47 ^g/_mol (C₂₂H₄₃NO). Both of these compounds have not been identified, because they are not in the Dictionary of Natural Product yet.