Actinomycetes abundantly present in soil are renowned for various secondary metabolites production with a wide range of use in diverse fields of life. Many reports are available on the use of actinomycetes for the regulation of pathogenic microbes in plants. For this purpose, this study used thirteen Actinomycetes strains, which were isolated from the fields of Piplan (district Mianwali) Punjab, Pakistan. However, out of thirteen, only seven were found active for the production of both primary and secondary screening. The active seven actinomycetes (AW1, AW2, AW3, AW4, AM1, AC1, and AC2) were separated for their microbial potential against pathogenic strains (Escherichia coli and Staphylococcus aureus) isolated from the same agriculture soil. About 42.85% of isolated actinomycetes (three out of seven) showed antagonistic properties against S. aureu and E. coli in primary screening. Thus, it was noticed that the three strains (AW3, AM1, and AC1) had great antimicrobial potential, which showed the most promising results. Whereas in Secondary screening only AC1 showed the best result with ethyl acetate extract as compared to ethanol, methanol, and chloroform. Hence, It was concluded that actinomycetes isolates had great potential for antibacterial activity and they can be used in agriculture industry for the regulation of plant pathogens.