Oyster mushrooms are recognized for their health-promoting phenolic compounds, which contribute to significant antioxidant properties. Efficient extraction and accurate quantification of these compounds are essential for advancing their application in nutraceuticals. This study aimed to develop and validate a microwave-assisted extraction (MAE) method for profiling phenolic compounds in a diverse selection of oyster mushrooms (Pleurotus spp.) sourced from multiple geographical regions. Using the Box-Behnken design and response surface methodology, three primary factors—solvent composition (0–30 % methanol in water), solvent-to-sample ratio (10:1–20:1 mL g−1), and temperature (40–70 °C)—were optimized. The optimal extraction conditions identified were pure water as a solvent, a 17.5:1 solvent-to-sample ratio, and a temperature of 44 °C, with a 10-min extraction time. The validated MAE method demonstrated high accuracy (>85 % recovery) and precision (CV <10 %), meeting analytical standards. Seven phenolic compounds—p-coumaric acid, t-cinnamic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, quercetin-3-glucoside, gallic acid, and vanillic acid—were effectively extracted and quantified using ultra-performance liquid chromatography with photodiode array detection. Application of this method across oyster mushroom samples from different geographical sources revealed significant variation in phenolic compound concentrations, with Pleurotus pulmonarius exhibiting the highest level of gallic acid at 433.90 ± 7.77 μg g−1 (DM) and Pleurotus ostreatus var. Florida showing the highest concentration of ρ-hydroxybenzaldehyde at 199.68 ± 1.06 μg g−1 (DM). This optimized MAE method proves to be a reliable tool for phenolic profiling in diverse oyster mushroom varieties, supporting potential applications in functional food, pharmaceutical, and nutraceutical industries.