Phenolic compounds, secondary metabolites in the herb of the endemic species Satureja horvatii Šilić (Lamiaceae), exhibit antioxidant activity, highlighting the medicinal value of this species. Natural deep eutectic solvents (NADES) represent a new generation of environmentally friendly and sustainable solvents, developed in accordance with green chemistry principles. These solvents are gaining attention as alternatives to conventional solvents. This study aimed to assess the effect of various NADES, combined with the green microwave-assisted extraction (MAE), on the preliminary chemical composition and antioxidant potential of extracts from S. horvatii herb. The effectiveness of NADES was also evaluated in comparison with conventional solvents, including ethanol and water. Four types of NADES were prepared through a combined heating–stirring technique, employing choline chloride, lactic acid, malic acid, glucose, and glycerol as components, in appropriate molar ratios. Six extracts of S. horvatii were produced through MAE, utilizing NADES, ethanol, and water as extraction media. Extraction was conducted in three 5-second cycles. Spectrophotometric techniques were used to determine the total phenolic content (TPC) and the total flavonoid content (TFC) of the extracts. The antioxidant potential of the extracts was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and the cupric reducing antioxidant capacity (CUPRAC) assay. The glycerol–lactic acid NADES enabled the extraction of the highest TPC, while the choline chloride–lactic acid and glycerol–lactic acid NADES exhibited the strongest antioxidant activity in the CUPRAC assay. The application of the MAE–ethanol combination yielded extracts with the highest TFC and the most potent DPPH radical scavenging activity. Lactic acid-based NADES represent an efficient and sustainable alternative to conventional solvents, providing extracts with TPC, TFC, and antioxidant activity, comparable to or exceeding those of ethanol and water.

Acknowledgments: This work was supported by the Provincial Secretariat for Higher Education and Scientific Research, Province of Vojvodina (Grant No. 142-451-3474/2023).