Abstract: The selection of natural environments, like soil with diverse climates, can increase the chances of discovering unique isolates and potentially novel compounds. The region of Aswan in Egypt, known for its geographical and climatic diversity, was investigated for actinomycete isolates. One particular isolate, designated as Streptomyces thermolilacinus AZHD22, demonstrated the production of bioactive substances with antimicrobial properties against microbes. The objectives of this work were to discover the generating actinomycete isolate, describe the bioactive molecule, and optimize the growth conditions for the synthesis of antimicrobials. The best conditions for antimicrobial production were observed in sterile starch-nitrate broth, incubated at 45 °C with an adjusted pH of 8 and 1% NaCl concentration. Glucose at 1% concentration was determined as the optimal carbon source, while peptone at 0.2% concentration provided the best nitrogen source. Additional components, such as 2.5 g/l K2HPO4·3H2O and 0.5 g/l MgSO4, were added to the broth media, followed by incubation in a shaker at 150 rpm for 9 days. The bioactive substances were extracted using ethyl acetate as the solvent, and purification revealed that fraction 9 exhibited potent antimicrobial activity against selected test microorganisms. Through characterizations including UV, IR, HNMR, and GC-Mass spectrum analysis, the bioactive compound was identified as (9,12-Octadecadienoic acid (Z, Z)-, methyl ester) with the chemical formula C19H34O2. The compound exhibited non-lethal effects on Vero cells up to a concentration of 250 μg/mL.