Objective: The study was aimed to assess the isolation method for Actinomycetes, understand the current status of the presence of antibiotics producing Actinomycetes in the soil sample of Hile and Jhapa, and evaluate the different methodology of testing antimicrobial activities. Methods: Actinomycetes were isolated from five soil samples of Hile and Jhapa on starch casein agar (SCA) media supplemented with amphotericin and rifampicin. Selected cultures were tested for antibiotic-producing properties in primary screening by placing isolates with the plate of test organisms. Antimicrobial activity was tested in secondary screening using agar well diffusion method from solvent extracts of potential Actinomycetes produced by submerged fermentation. Results: Out of seven isolates of Actinomycetes from five tested soil samples, two isolates showed some antimicrobial activity against Gram-positive test organisms viz Staphylococcus spp. and Bacillus spp. A larger zone of inhibition was shown by actinomycetes sample AG4 against Staphylococcus spp when compared with other test organisms. Conclusion: The findings show the possibility of antimicrobial activity of the Actinomycetes isolates in the soil of Hile and Jhapa and highlight the necessity of extensive studies for the isolation and purification of bioactive metabolites. Molecular characterization analysis of isolated Actinomycetes spp would help discover novel compounds of commercial value.