Calotropis gigantea is very unique plant which belongs to family Apocynaceae. According to Shivpuran the flowers are very much liked by lord Shiva even it is also one of the major parts of nine astrological plants but it is found to have many medicinal values. In the present study, a sensitive, reliable and rapid ultra-performance high resolution liquid chromatography method was established for determination of Deoxy streptomycin, Protorifamycine-I, Citrulline, 4-Trimethylammoniobutanal, cystamine, 6-hydroxy-2-hexynoic acid, Carnitine, D-Arginine, Choline, Pantoic acid, Betaxol, Madecassic acid, Avocadynone acetate, 6-deoxyerythronolide-B, O-Acetylserine, 4,6-Diamino-5-formidopyrimidine, Dihydrorobinetin, Barbituric acid, 5-ethyl-5-(2- hydroxyethyl), Idebenone Metabolite isolated from the leaves extract of Calotropis gigantea. Chromatographic separation was accomplished on a C18 column with a multiple-step gradient elution using water and acetonitrile (95: 5) with 30 minutes acquisition time as mobile phase. Antifungal activities were estimated by the food poison technique whereas, agar disc diffusion method was used for antibacterial activity. The results showed that phytoconstituents were separated and identified by HR-LCMS from the leaves of Calotropis gigantean revealed antimicrobial activities against Fusarium oxysporum,Colletotrichum capsici and Xanthomonus axonopodis.