The study is aimed at process of isolation, purification and characterization of actinomycetes species from soil which exhibits antimicrobial property versus 8 chosen pathogenic strains. The soil samples collected from different habitats present in Secunderabad, India. The collected samples were subjected to serial dilution and are plated on agar media containing actinomycetes isolates. Screening, purification and storage of potential colonies in glycerol stock solution is carried out followed by characterization of isolates biochemically and morphologically is carried out. The extraction process of isolates is carried out to produce antibacterial compound and evaluation process is conducted by Minimum inhibitory concentration method. A total of 6 isolates of actinomycete species were tested for antagonistic property vs 8 pathogenic microbes. S-4 among isolates found to be very active and activity of the isolate is further increased by the influence of UV mutation. The strain AM2 exhibited maximum antibiotic producing property which is 4.9 mg/ml and it is referred to as S-4 mutant strain. S-4 mutant strain is further used to carry out fermentation related studies.