Barleria prionitis L. leaf ethanol extract has the potential as an antibiotic. To obtain a simpler compound from ethanol extract, it is necessary to fractionation the antibiotic of landep leaves using hexane and ethyl acetate gradient and to conduct antibiotic testing using Kirby Bauer method on Staphylococcus aureus and Escherichia coli bacteria. Solvent gradients that have the potential as antibiotics include hexaneethylacetate (1: 9) and (2: 8). Analysis of the compounds responsible for the potential antibiotic activity of each fraction using GCMS (Gass Chromathography Mass Spectrum) analysis instruments. The results of the analysis of the hexane-ethylacetate (1: 9) fraction produced 70% similarity of 2 (3H) -Furanone, dihydro- (CAS) Butyrolactone. The solvent gradient of hexane-ethylacetate (2: 8) produced 89% similarity to 6.8-Dioxabicyclo (3.2.1) Octan-2. Beta.-Ol-3.3-D2