The purpose of this research is to know the composition of fraction oil kaffir lime which is consist as antibacterial activity E.coli. This research uses kaffir lime oil from a branch for fractional distillation use PiloDist 104-VTU (number the stages 120), and reflux ratio 20/10 with 5 mbar pressure. Oil kaffir lime composition analysis use GC-MS (type Shimadzu QP 2010S) with a gas helium on speed 3mL/minute. Antibacterial activity assay use agar well diffusion assay which does in three concentration they are 500 µL/mL, 300 µL/mL, 100 µL/mL. The result of oil fraction kaffir lime has five fraction oil based on boiling point interval, they are A fraction oil (63,00 – 70,010 C), B fraction (71,30 – 70,800 C), C fraction (74,50 – 74,200 C), D fraction (74,20 – 74,000 C) and E fraction (72,90 – 91,100 C). The result of this research obtained five oil fraction they are monoterpene oxygenated (MO) except A oil fraction with monoterpene hydrocarbon composition (MH) 12,1%. The main component of a fraction which MO compound they are citronella, linalool and isopulegol, while in MH compound they are sabine, β-pinen, β-micren and limonene. The result of antibacterial activity assay obtained on highest concentration it is 500 µL/mL. Antibacterial activity assay also depends on fraction composition with higher composition of MO. The highest MO components of oil fraction on C oil fraction which has MO component such as citronella 74,94%; linalool 20,13%; and isopulegol 3,08%.