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Astragalus membranaceus (Huang Qi) is a traditional medicinal plant known for its antioxidant, antibacterial, and anti-inflammatory properties. To evaluate the biological potential of in vitro-derived biomass, we established and compared three tissue types of A. membranaceus: cell suspension (CS), adventitious roots (AR), and acclimated plants (AP). Culture conditions were optimized, with CS exhibiting maximal proliferation at a 2% inoculation density in liquid MS medium with 1.0 mg/L 2,4-D and 0.5 mg/L IBA, while AR development and lateral root branching were maximized with 2.0–3.0 mg/L IBA. Total phenolic content (TPC) and total flavonoid content (TFC) were quantified using colorimetric assays, and antioxidant activity was assessed via DPPH radical scavenging and reducing power methods. Antibacterial activity against Escherichia coli and Staphylococcus aureus was evaluated using zone of inhibition assays, while anti-inflammatory effects were determined by nitric oxide (NO) production and expression of pro-inflammatory cytokine genes in lipopolysaccharide-stimulated RAW264.7 macrophages. The results showed that AP contained the highest TPC and TFC, corresponding with the strongest antioxidant activity. Moreover, CS exhibited superior antibacterial activity against S. aureus and significantly reduced NO production and expression of inflammatory markers (iNOS, TNF-α, IL-6, and IL-1β). All extracts were non-cytotoxic up to 200 μg/ mL. These findings highlight distinct bioactivity profiles among biomass types, with AP suited for antioxidant applications and CS more effective for anti-inflammatory and antimicrobial activities. This work provides foundational insight into the targeted application and optimization of A. membranaceus in pharmaceutical and functional product development.

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