The objective of this study was to extract and purity the antibacterial agent from the fermented milk with Lactobacillus helveticus CH-1. The extraction and purification of antibacterial agent from the Lb. helveticus fermented milk were carried out by methanol extraction, acetone extraction, Sephadex G-200 gel filteration and thin layer chromatography and the results were as followings. The antibacterial activity of methanol-acetone extraction showed antibacterial activity against test organisms, B. subtilis, E. coli, Pseu. fluorescens, Sal. typhimurium, Shi. flexneri, and Sta. aureus. Sephadex G-200 gel chromatography showed only antibacterial activity from 33 to 37th fractions of 60 fractions. The agent purified from TLC plate confirmed the antibacterial activity by the means of bioautography.