ABSTRACTThree purified proteins G-I, G-II and G-III were obtained from Bacillus sp using the techniques of DEAE Sepharose Fast Flow Chromatography, filtration chromatography. anion exchange chromatography, molecular determination by SDS page gel electrophoresis (SDS-PAGE). The purity of G-I, G-II and G-III was measured by DEAE Sepharose Fast Flow Chromatography. G-I, G-II and G-III were measured by SDS-PAGE to have molecular weights of 14.4 k Da and 94.4 k Da, and three bands appear in the molecular weights of 46 k Da band 35 k Da and 32 k Da band respectively. The amino acid analysis of purified compound G-III was determined because greater antimicrobial activity of G-III compound. The amino acid analysis using technique of automatic amino acid analyzer (Shimatzu-High performance liquid chromatography LC 4A) 20 µl of the purified sample was injected into single column and analyzed using sodium buffer system. G-I, G-II and G-III inhibited the clinical pathogens Staphylococcus aureus and Pseudomonas aeruginosa. By antimicrobial activity of Agar well diffusion assay. Test the clinical pathogens shoe the greater activity of G-III and Zone of inhibition various concentration of (25 µl, 50 µl, 75 µl, 100 µl). G-III shows the maximum results of 18.2mm and 15.4mm against Staphylococcus aureus and Pseudomonas aeruginosa. Keywords: Bacillus sp; protein; purification; in vitro antimicrobial activity.