To be dyed with cationic Berberine, the cotton fiber was modified with reactive anionic agent. The Berberine, a natural cationic colorant of pure yellow, is the major component of Amur Cork tree extract and also can be employed as a natural antimicrobial agent due to its characteristic of cationic quaternary ammonium salt. By LC/MS analysis, it became obvious that the Berberine was contained in Amur Cork tree extract as a major color component. The adsorption of the Berberine on the cotton fabrics pretreated with the anionic agent was greatly increased comparing to that of untreated fabric. Because the anionic agent was colorless, it did not cause unintended color change of the dyeings. The dyed fabric with the Berberine has strong antimicrobial activity showing 99.5% of reduction of bacteria against Staphylococcu aureus.