Polyphenols were extracted from Thuja occidentalis L. bark and characterized in terms of phytochemical composition and biological activity. The optimization process investigated the impacts of extraction duration, ultrasound amplitude, and ethanol concentration on the total phenolic content in the extract. The antioxidant capacity was examined using DPPH and ABTS assays, and anti-enzyme activity against alpha-glucosidase, alpha-amylase, lipase, acetylcholinesterase, and tyrosinase was determined. The experimental model revealed optimal extraction parameters: a hydroethanolic solvent with 44% v:v ethanol, a 15-minute extraction time, and a 40% ultrasound amplitude. These parameters were validated and confirmed by reaching the target Total Phenolic Content (TPC) predicted by MODDE software. The resulting lyophilized extract exhibited a high polyphenolic content (161.84 ± 23.48 mg GAE/g dry extract) and demonstrated strong antioxidant properties. Notably, it showed significant inhibition of alpha-glucosidase and alpha-amylase, suggesting potential antidiabetic effects, though its inhibition of tyrosinase activity was relatively weak. These findings provide a foundation for further investigations to confirm the qualitative and quantitative presence of specific polyphenolic metabolites in the extract and elucidate the mechanisms underlying its bioactive properties
