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The high‐speed countercurrent chromatography technique was employed to isolate two major compounds from the leaf essential oil of Eugenia uniflora L. (Myrtaceae). The volatile extract of Eugenia uniflora obtained by hydrodistillation was analyzed by gas chromatography coupled with flame ionization detection and gas chromatography coupled with mass spectrometry and shown to be almost exclusively constituted by sesquiterpenes, in which (+/‐)‐selina‐1,3,7(11)‐trien‐8‐one (A) and (+/‐)‐selina‐1,3,7(11)‐trien‐8‐one epoxide (B) were largely predominant. These two major compounds were separated by submitting the crude essential oil to countercurrent chromatography using hexane/acetonitrile 1:1 v/v as the solvent system, running in both the tail‐to‐head (mode I) and head‐to‐tail (mode II) modes. Upon running in mode I, a single application of the crude oil (800 mg) in the separation equipment coil afforded 78.2 mg of B (> 97.5% purity). Running in mode II produced 24.7 mg of A (92.5% purity) and 46.3 mg (93.1% purity) of B. The purity of each compound was determined by GC. For the first time, this technique was shown to be efficient to separate sesquiterpenes that are structurally very similar, affording high‐purity products in a short time with significant solvent savings.