In the current study, the effect of glycerol -as a green solvent- addition into solvent mixture (50:50 ethanol:distilled water), on some biofunctional properties of Origanum onites L. was investigated. Response surface methodology (RSM) was used to detect the optimum conditions for the extraction process. Three variables namely glycerol concentration (X-1: 1-9 g), extraction temperature (X-2: 25-75 degrees C) and time (X-3:10-30 min) were selected and also total phenolic content, total flavonoid content, antioxidant capacity and antiradical activity of O. onites extracts were determined. Analysis of variance (ANOVA) showed that glycerol incorporation significantly increased the total phenolic content and antioxidant activity (p < 0.05) of the samples. Maximum levels to obtain the highest bioactive properties (highest total phenolic content and antioxidant activity) were determined as to be 9 g of glycerol addition for the extraction conditions as 45.4 degrees C and 75 min. This study reports the effect of glycerol on bioactive properties of O. onites and suggests that glycerol can be used to produce hydroalcoholic extracts having higher bioactivity from Origanum genus. (C) 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).