Jateorhiza macrantha is a known medicinal plant used in ethno medicine for the treatment of diarrhea, cough, boils and ulcer. This study is aimed at evaluating the antibacterial activity of chromatography fractions from the aqueous ethanol extract of the leaves of Jateorhiza macrantha as a preliminary step towards isolation and characterization of its anti-bacterial constituent(s). The aqueous ethanol extract (AEE) was obtained by exhaustive cold maceration in 70% aqueous ethanol and further fractionated using vacuum liquid chromatography (VLC) techniques with a mobile phase gradient of n-hexane:chloroform:methanol (4:0:0, 3:1:0, 2:2:0, 1:3:0, 0:4:0, 0:3:1, 0:2:2, 0:1:3, and 0:0:4 v/v/v) into three fractions: non-polar (NPF1, eluted with n-hexane:chloroform:methanol 4:0:0, 3:1:0, 2:2:0 v/v/v), moderately polar (NPF2, eluted with n-hexane:chloroform:methanol 1:3:0, 0:4:0, 0:3:1, 0:2:2, 0:1:3 v/v/v) and polar (NPF3, eluted with n-hexane:chloroform:methanol 0:0:4 v/v/v). Antibacterial activity was evaluated using the agar well diffusion method against clinical isolates of pathogenic bacteria: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pnuemoniae, and Salmonella typhi and the Minimum Inhibition concentration (MIC) extrapolated following the graphical approach. Functional group investigation was done using infra red spectroscopy. Three chromatography fractions NPF-1 –NPF3 obtained had MIC range of 5.0 mg/ml > MIC <0.26 mg/ml. NPF3 was relatively pure showing a single spot on TLC and its IR spectra are evidence for OH, N-H, C-O, C=C, and C-H stretching and deformation vibration frequencies typical of amino derivative of a lactone glycoside. The observed antibacterial activity justifies the ethno medicinal uses of Jateorhiza macrantha.