T HE utilization of ultrasonic waves in textile coloration processing possesses many hopeful advantages. Conventional method in dyeing processes consumes a large amount of dye and thermal energy. The dyeing processing is exclusively carried out at higher temperatures. Ultrasonic dyeing is a novel technique to save time, cost, energy and provides high value of dye uptake. The aim of this study was to investigate the dyeing behavior of pre-treated wool fibers with chitosan, then dyed with rhubarb as natural dye by conventional (Con) and ultrasonic (US) methods. Factors affecting the dye extraction and dyeing processes such as dye concentration, temperature, time as well as pH was studied. The results indicated that the pre-treated wool fibers with chitosan get higher color strength (K/S), and all over color fastness properties values if it compared with the untreated fibers. Pre-treated wool fibers can be dyed at lower temperatures in ultrasonic method as compared with conventional method. In this study the antimicrobial activity with some kinds of Bacteria and Fungi were verified. The results demonstrated that the pre-treated fibers exhibit higher inhibition percent than the untreated one. As a result, the utilization of ultrasonic waves in dyeing procedure helps to decrease the dyeing temperature, thereby reducing energy consumption and maintaining the extracted and dyeing quality.