Antibacterial secondary metabolite is a bioactive compound like antibiotic that can be considered a substance, produced by one microorganism, which in low concentration inhibits the growth of other microorganisms. Actinomycetes, slow-growing gram-positive bacteria, are the major sources of bioactive compounds. This study aimed to screen and identify antibacterial secondary metabolite-producing actinomycetes by sequencing the 16S rRNA method (molecular identification) from the soils of the mountain region of eastern Nepal. Starch casein agar (SCA) medium was used for the isolation of actinomycetes which were confirmed by primary screening and secondary screening. Identification of presumptive genera was done based on macroscopic, microscopic, and biochemical characteristics and confirmed by sequencing their 16S rRNA genes. The antibacterial compound was produced by culturing the potential isolate in starch casein broth. Using organic solvents such as ethyl acetate, n-butanol, chloroform, dichloromethane, and methanol, the chemical was recovered from the fermented broth. TLC performed the antibacterial substance characterization. Only 9 (13.6%) of the 66 actinomycetes isolates showed antibacterial activity against test microorganisms. Only one of the nine isolates, M3, had antibacterial activity in primary screening against gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli, Salmonella Typhi, Shigella spp., and Pseudomonas aeruginosa) test bacteria. M3 was chosen for secondary screening due to its strong antibacterial activity. The minimum inhibitory concentration (MIC) of crude antibacterial substances was found to be 2.5 mg/mL against test organisms. According to the TLC chromatogram, the isolate produced only one compound with an Rf value of 0.81, completely distinct from the spot formed by gentamicin (standard), which had an Rf value of 0.89. The isolates were considered Streptomyces spp., a distinct taxonomic group based on characterization by macroscopic, microscopic, biochemical, physiological, and molecular techniques. This study concluded that Mountain regions are the reservoir of antibiotic- producing actinomycetes. Streptomyces is the most common genus.