Acacia hybrid is a fast-growing tree species which plants in the forest plantation for energy in Southeast Asia, including Thailand. The efficient method for rapid propagation for forest plantation is tissue culture technology. A sterilization is one of the most important steps in tissue culture. The effects of type, concentration, and duration of disinfectants were examined. Axillary bud was used as explant for in vitro propagation. The procedures for sterilization were conducted six treatments and cultured in ½MS for 4 weeks. There was significantly different for all treatments in the percentage of survival, contamination, dead explants, and growth performance (P<0.05). The optimal sterilization procedure was rinsed in running water for 5 minutes +20% dishwashing liquids+70% (v/v) Ethanol for 30 seconds + 0.1% HgCl2 for 10 minutes and rinsing with sterile water for 4 times. The results showed 13.50±9.26% contaminated explants and 86.50±9.26% survived explants. The shoot length, leaf length and leaf number were 3.40±0.20 cm., 5.04±0.70 cm. and 3.3±0.48 no./explant, respectively. Axillary bud from sterilized explant was transferred to MS medium supplemented with 30 g/l sucrose+0.6 mg/l BAP+0.6 mg/l NAA+0.3 mg/l PVP for shoot multiplication. The maximum number of shoots after 8 weeks was 10.5±1.06 shoots/explant. Each microshoot was elongated and rooted on MS basal medium without plant growth regulators.