Objective: The study’s goal was to evaluate the isolation method for Actinomycetes, identify the current state of the existence of antibiotic-producing Actinomycetes in soil samples from Dharan and Belbari, and evaluate various antimicrobial activity testing methodologies. Methods: Actinomycetes were recovered from five soil samples from Dharan and Belbari using starch casein agar (SCA) media containing amphotericin and rifampicin. In primary screening, isolates were placed on a plate of test organisms to see if they produced antibiotics. Antimicrobial activity was assessed in secondary screening utilizing the agar well diffusion method with solvent extracts of putative Actinomycetes prepared by submerged fermentation. Results: Four Actinomycetes isolates from five soil samples shown antibiotic efficacy against Gram-positive pathogens, including Staphylococcus and Bacillus. Actinomycetes sample AG10 demonstrated a greater zone of inhibition against Staphylococcus spp. when compared to other test organisms. Conclusion: The study suggests that Actinomycetes isolates in Belbari and Dharan may have antibiotic potential. Further research is needed to isolate and purify bioactive compounds from these soils. Molecular characterization of isolated Actinomycetes spp. would aid in the discovery of novel substances with commercial value.