Considering the vast potentiality of Tinospora cardifolia as a source for antimicrobial drugs with reference to antibacterial, antifungal and anticandida agents, a systematic investigation was undertaken to screen 17 samples of T. cardifolia accessions collected from different Districts of Himachal Pradesh for its activity against gram positive and gram negative bacteria, Fungus and Candida strains. The zone of inhibition varied with the plant extract, the solvent used for extraction and the organism tested. Acetonic leaf extract was found to be more potent being capable of exerting significant inhibitory activity than other extracts. Highest antimicrobial activity was observed with the acetonic leaf extract of T. cardifolia collected from Baijnath, District Kangra. FT-IR analysis of the acetonic leaf extract revealed the presence of different functional groups at wave numbers corresponding to the functional groups of a number of compounds including Berberine. Thin layer chromatography and HPLC of acetonic leaf extract of T. cardifolia indicated alkaloid as a major active compound which was further confirmed to be Berberine. Active component was isolated and purified by recrysyallization method and tested for its antibacterial activity. The isolated compound was characterized by HPLC, LCMS, GCMS, H1 and C13 NMR. HPLC peak of isolated Berberine at retention time 4.024 mins was observed which was same as that of standard Berberine. Molar mass observed by LCMS and GCMS was 336.1 which were same as that of standard Berberine. NMR also confirms the presence of 18 H atoms as that present in standard Berberine.