Aim of the present study is to isolate and identify promising antimicrobial metabolite producing Streptomyces strain from Datura stramonium. Physiological, biochemical and 16S rRNA studies strongly suggested that this isolate belonged to Streptomyces spp and ability to produce enzymes such as amylase, lipase and catalase. Maximum biological activity was obtained on Modified Nutrient Glucose Agar (MNGA) medium. Preliminary screening revealed that the isolate was found to be active against bacteria and fungi. Isolate showed activity against bacteria such as Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and fungi such as Aspergillus niger, Trycophyton rubrum and Aspergillus flavus. The antibacterial substances were extracted using methanol from MNGA medium in which isolate had grown for five days at 28°C. The antimicrobial activity was assessed using broth microdilution technique. The lowest Minimum Inhibitory Concentration of methanol fractions of Streptomyces spp. Loyola UGC against B. subtilis, S. aureus was 250 mg/ml and against Aspergillus flavus was 61.5 mg/ml. The growth parameters such as carbon source, Nitrogen source, Inoculation period, pH and temperature were optimized. Nutritional and cultural conditions for the production of antimicrobial metabolite by this organism under shake-flask conditions have been studied. HPLC analysis of methanol extract of actinomycets showed the presence of Hyoscyamine and scopolamine.