| Summary: | <i>Picnomon acarna</i> (L.) Cass. is a Mediterranean medicinal plant with limited phytochemical and bioactivity characterisation. In this study, methanolic extracts obtained by maceration (MAC), Soxhlet (SOE), and ultrasound-assisted extraction (UAE) were comparatively investigated to determine their phytochemical composition and biological potential. Liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) analysis identified and quantified 24 phenolic compounds, with hesperidin, chlorogenic acid, and hyperoside as the dominant constituents. The maceration extract exhibited the highest total phenolic content (29.06 mg GAE/g extract) and showed superior antioxidant performance across six complementary assays [2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), ferric reducing antioxidant power (FRAP), phosphomolybdenum, and ferrous-ion chelation), reflected by the highest relative antioxidant capacity index (RACI = 0.93). Enzyme inhibition assays revealed extraction-dependent activity patterns: Soxhlet and ultrasound extracts demonstrated stronger acetylcholinesterase inhibition (IC<sub>50</sub> ≈ 1.23 mg/mL), while Soxhlet extract showed the most potent tyrosinase (AChE) inhibition (IC<sub>50</sub> = 1.48 mg/mL). α-Amylase inhibition was comparable among extracts (IC<sub>50</sub> = 1.90–2.03 mg/mL). Pearson correlation analysis indicated strong relationships between major phenolics and antioxidant activity. Molecular docking further supported these findings, showing favourable binding affinities of hesperidin, hyperoside, and chlorogenic acid toward α-amylase and acetylcholinesterase, while only chlorogenic acid and hyperoside demonstrated favourable interactions with tyrosinase-related protein-1 (TYRP1), whereas hesperidin did not exhibit a meaningful binding affinity. Overall, the results demonstrate that the extraction strategy significantly influences the phenolic composition and multi-target bioactivity of <i>P. acarna</i>, highlighting its potential as a source of natural antioxidant and enzyme-modulating compounds.
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