Momordica Charantia (Cucurbitaceae) is commonly known as bitter guard in English and karela in Hindi. The fruit has claimed to contain charantin, steroidal saponin, momordicosides, carbohydrate, mineral matters, ascorbic acid, alkaloids, glucoside, etc. Earlier claim shows that the plant used as stomachic, carminative, tonic, antipyretic, antidiabetic, in rheumatoid arthritis and gout. The present investigation was carried out to isolate, purify and characterize Charantin from fruit of Momordica Charantia Linn. The isolated charantin was further characterized with the help of Ultraviolet Spectroscopy, Thin Layer Chromatography, Fourier Transform Infra Red Spectroscopy, Mass Spectroscopy, Proton- Nuclear Magnetic Resonance Spectroscopy confirmed the identification. The antibacterial activity of charantin was tested by using Agar Diffusion (Cup Plate) method. The minimum inhibitory concentration (MIC) of crude extracts were determined for various organism which was 0.2 mg/ml. The present studies confirm better antimicrobial activity of Charantin when compared with standard, against bacterial species such as gram positive (Bacillus subtilis), gram negative (Pseudomonas aeruginosa) and fungal strains (Saccharomyces cerevisiae).