Oregano (Poliomintha longiflora) essential oil (Ooil) is a product of high commercial value and many applications, including chemotherapy. Aiming to achieve the best use of this resource, the present study focuses on the characterization of separated fractions of Ooil by fractional vacuum distillation at low pressure. Four fractions (F1–F4) and undistilled oil (Unoil) were separated from Ooil and analyzed for their chemical composition and biological activities, such as antioxidant and antimicrobial activities. Gas chromatography–mass spectrometry shows differences in the composition among the fractions and Ooil. The amount of monoterpenes oxygenated (MO), sesquiterpenes hydrocarbon (SeH) and monoterpenes hydrocarbon (MH) varied between the fractions in ranges of 1.51–68.08, 3.31–25.12 and 1.91–97.75%, respectively. The major concentrations of MO and SeH were observed in F4 and Unoil. On the other hand, the highest concentrations of MH were found in F1 and F2, while the lowest were in F4 and Unoil. These results were correlated with the biological activity. Free-radical scavenging activity varied among fractions, with F4 and Unoil showing the highest activity. The antimicrobial test showed that F4 and Unoil had the highest activity in almost all cases. The correlation between the variables studied in the different fractions allows the definition of the particular properties for each one of them.