Research on Actinomycetes has proven highly beneficial to society, particularly in the development of product-oriented solutions for disease cures through the utilization of their compounds, commonly known as drugs. This study focused on screening and identifying actinomycete strains derived from the root nodules of Cissus quadrangularis and Alpinia officinarum cultivated in Chennai. The research began with serial dilution and enumeration of soil samples, followed by screening and isolation procedures aimed at discovering novel species. The study yielded 13 Actinomycete strains identified based on Gram staining, colony morphology, and biochemical tests, all of which exhibited Gram-positive characteristics with varying colony morphologies and biochemical profiles. Primary screening determined antibacterial isolates and identified the range of microorganisms sensitive to the antibiotics. A secondary screening was conducted to validate the results. Both qualitative and quantitative approaches were employed to determine the sensitivity of microorganisms to potential antibiotics. The qualitative method determined the range of sensitive microorganisms, while the quantitative approach provided insights into the expected antibiotic yield when the organisms were cultured in ISP2 broth. All experimental measurements were conducted in triplicate, and the results were expressed as averages of three analyses. Among the isolates, I5, MS1005F, and MS1005E demonstrated high activity, while MS100E exhibited relatively lower activity against pathogenic bacterial strains. Notably, isolate I5 displayed the highest activity against MS1005F.