Actinomycetes are one of the most important groups that produce useful secondary metabolites. They play a great role in pharmaceutical and industrial uses. The search for antibiotic producing soil actinomycetes to inhibit the growth of pathogenic microorganisms has become widespread due to the need for newer antibiotics. The present work was aimed to isolate soil actinomycetes from pinus tree rhizosphere from Doddabetta, Western Ghats, Tamil Nadu, India. Thirty one actinomycetes were isolated based on heterogeneity and stability in subculturing; they were screened against 5 Gram positive and 7 Gram negative bacteria in an in vitro antagonism assay. In the preliminary screening, out of 31 isolates, 12.09% showed good antagonistic activity; 25.08% showed moderate activity; 19.35% showed weak activity and 41.93% showed no activity against the tested bacteria. Among the isolates tested, DPR20 showed good antibacterial activity against both Gram positive and Gram negative bacteria. The antibacterial crude secondary metabolites were extracted using ethyl acetate from micromonospora medium (M3) in which DPR20 was grown for twelve days at 30ºC. The lowest MIC value of ethyl acetate extract against Bacillus subtilis and Staphylococcus epidermis was 31.25µg/ml. The active isolate was identified as Streptomyces collinus DPR20 based on morphology, physiology, scanning electron microscopy and 16S rRNA gene sequence analysis.