The separation of banana fibers from the pseudostem layers and the properties of two Thai banana cultivars, Musa acuminata (Kluai Hom) and Musa sapientum L. (Kluai Namwa) were investigated. The pseudostems were divided into three layers: outer (layer 1), middle (layer 2), and inner (layer 3). Fibers were extracted using a semi-automatic machine and subjected to chemical treatments. Results showed that layer 3 of Musa sapientum L. pseudostems yielded the highest amount of fibers with the lightest color. Fibers from layer 1 of Musa sapientum L. exhibited the highest tensile strength (606.90 g/denier) and elongation at break (9.54 %), higher than in all layers of Musa acuminata. SEM analysis revealed that chemically treated Musa acuminata fibers were less separated and broken than Musa sapientum L. fibers because the chemicals disrupted the lignocellulose bonds. Cross-sectional images showed larger lumens and more pronounced fiber group separation in Musa sapientum L. The outer layer of Musa sapientum L. pseudostem provided fibers with superior strength and elongation, demonstrating high potential as a sustainable raw material for the textile industry.