Purpose <p>We developed a novel chitosan-based microencapsulation system for <i>Mitragyna speciosa</i> alkaloids to enhance their therapeutic potential in Alzheimer’s disease (AD) treatment, focusing on cholinesterase inhibition and antioxidant properties.</p> Methods <p>Three <i>M. speciosa</i> strains were fractionated and evaluated for anti-cholinesterase activity, antioxidant properties, and mitragynine content using HPLC analysis. The optimal fraction was encapsulated in chitosan microparticles using ionic gelation. The formulation was characterized for morphology, particle size, zeta potential, and encapsulation efficiency. Release kinetics and maintained biological activity were assessed through simulated gastrointestinal digestion.</p> Results <p>The alkaloid fraction from green-veined variety (MAG) exhibited superior acetylcholinesterase (IC₅₀: 70.17 ± 0.98&#xa0;µg/mL) and butyrylcholinesterase inhibition (IC₅₀: 54.39 ± 5.43&#xa0;µg/mL), correlating with its highest mitragynine content. MAG-loaded chitosan microparticles demonstrated optimal characteristics with uniform spherical morphology (diameter: 665.9 ± 16.5&#xa0;nm, PDI: 0.369 ± 0.006), high stability (zeta potential: +57.11 ± 3.15 mV), and efficient encapsulation (72.60 ± 0.25%). The formulation showed controlled release under simulated gastrointestinal conditions while maintaining anti-cholinesterase activity.</p> Conclusions <p>This study presents an effective microencapsulation strategy for enhancing the therapeutic potential of <i>M. speciosa</i> alkaloids. The optimized formulation demonstrates improved stability and controlled release properties, offering a promising approach for AD treatment through sustained biological activities.</p>