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This research aimed to optimize pressure (10–20 MPa) and temperature (45–60 °C) conditions for supercritical fluid extraction (SFE) of Makwaen pepper (Zanthoxylum myriacanthum) extract (ME) in comparison to conventional hydro-distillation extraction. Various quality parameters, including yield, total phenolic compounds, antioxidants, and antimicrobial activities of the extracts, were assessed and optimized using a central composite design. The optimal SFE conditions were found to be 20 MPa at 60 °C, which resulted in the highest yield (19%) and a total phenolic compound content of 31.54 mg GAE/mL extract. IC50 values for DPPH and ABTS assays were determined to be 26.06 and 19.90 μg/mL extract, respectively. Overall, the ME obtained through SFE exhibited significantly better physicochemical and antioxidant properties compared to ME obtained through hydro-distillation extraction. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that beta-pinene was the major component in the ME obtained through SFE (23.10%), followed by d-limonene, alpha-pinene, and terpinen-4-ol at concentrations of 16.08, 7.47, and 6.34%, respectively. On the other hand, the hydro-distillation-extracted ME showed stronger antimicrobial properties than the SFE-extracted ME. These findings suggest that both SFE and hydro-distillation have the potential for extracting Makwaen pepper, depending on the intended purpose of use.