The purpose of this research is to evaluate the antibacterial fractions’ activity of Zingiber cassumunar Roxb. rhizomes against both Bacillus cereus bacteria and Klebsiella pneumoniae and to identify the most active fraction of them. The rhizome’s powder was macerated with 96% ethanol, then the resulting extract was evaporated and fractionated by vacuum liquid chromatography with comparation of hexane : ethyl acetate as eluent. The antibacterial activities were evaluated using diffusion method and then the most active fraction was evaluated by inhibition zone. The most active fraction was evaluated for Minimum Inhibitory Concentration (MIC) and its’ equivalent value compared to amoxicillin. Then, it was identified with Gas Chromatography - Mass Spectrometry (GC-MS). The F fraction showed the most active antibacterial fraction against two testing bacteria. The F fraction had MIC 0.075% against two testing bacteria. The F fraction antibacterial activity value was 5.864x10-3% compared to amoxicillin for Bacillus cereus and 3.094x10-3% for Klebsiella pneumoniae. The GC-MS result showed that the F fraction contained phenylbutenoid compounds and phenylbutenoid dimer compounds.