The exploration of novel therapeutic agents and other bioactive secondary metabolite from Streptomyces species, for possible agricultural farming, pharmaceutical and industrialized applications, has been, and still is, essential. The existing studies were aimed with biologically potential Streptomyces species and its antagonistic activity against dreadful microorganisms. Totally, morphological three different actinomycetes were selected from the fertile agricultural lands. Among the three, the isolate SA4 exhibited significant antimicrobial and anti-nematicidal activity towards selected microbial pathogens such as E Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Salmonella typhi, Bacillus subtilis, Proteus vulgaris, Shigella flexineri, Candida albicans, and Fusarium sp. The prospective strain SA4 was identified as Streptomyces cuspidosporus. The isolate SA4 optimized for secondary metabolites production with International Streptomyces project 4 (ISP 4) medium, pH 7.0 at 37 °C for 14 days. Gas Chromatography-Mass spectrometry (GCMS) analysis of strain SA4 bioactive extract publicized the existence of 1,2-Benzenedicarboxylic acid, bis(2-Methylpropyl) ester compound and occupied by high peak area and its possessed significant biological properties.