This study was conducted to determine the Antimicrobial Susceptibility Test (AST) of the Mangosteen fruit ethanol extract (Garcinia mangostana Linn) against Escherichia coli (E. coli) bacteria. The study used an experimental research design and made different concentrations of  Mangosteen fruit ethanol extract, with concentrations consisting of: Tube 1, 100% (no distilled water), Tube 2, 75% (250 uL [0.25 mL] of  distilled water), Tube 3, 50% (500 uL [0.5 mL] of distilled water), and Tube 4,  25% (750uL [0.75 mL] of distilled water). The positive control contains the antibiotic (Gentamicin), and negative control which is made of distilled water.  The researchers used a two-way analysis of variance (ANOVA) as statistical analysis. The computed P-value in treatments is  <0.0001 that indicates that there is a significant difference in the zone of inhibition of E. coli against various concentrations of Mangosteen Fruit  Ethanol Extract. While the computed P-value in trials is  1.0000 that indicates that there is no significant difference in the zone of inhibition of E. coli against various concentrations of Mangosteen Fruit  Ethanol Extract. It is therefore concluded that the different concentrations of Mangosteen Fruit  Ethanol Extract has no effect in inhibiting  the growth of E. coli.  Meanwhile when it comes to the effectiveness of concentrations (100%,75%,50%,25% of  Mangosteen Fruit  Ethanol Extract)  in the  inhibitory zone for antimicrobial E. coli, the result shows that it is ≤ 12 hence all of the concentration falls under Resistant category on the Kirby-Bauer Method.