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The tropical plant Mitragyna speciosa (Korth.) Havil is native to Southeast Asia, including Malaysia, Indonesia, and Thailand. Studies have identified advantageous biological attributes in the plant, such as antioxidant, anti-inflammatory, and antimicrobial, which are vital during wound healing. The current study aims to investigate the in vitro wound healing capacities of methanol crude extract and fractions of Mitragyna speciosa leaves on 3T3 fibroblast and human umbilical vein endothelial cells (HUVECs). The wound healing abilities of the extracts obtained were then assessed through cytotoxicity, scratch, and tube formation assays. The methanol crude extract and dichloromethane and ethyl acetate fractions were non-toxic against the 3T3 fibroblast cells and HUVECs at concentrations below 50 μg/mL. The scratch assay revealed that the ethyl acetate and dichloromethane fractions recorded better migration activities at low concentrations, 1.56 and 3.13 μg/mL. Furthermore, the ethyl acetate fraction recorded the highest branching length in the tube formation assay at all concentrations, thus demonstrating a strong potential for angiogenesis promotion. This study also demonstrated that targeted secondary metabolites, such as flavonoids, phenolics, and fatty acids, might possess therapeutic attributes, including anti-inflammatory, antibacterial, and antioxidant, as they are responsible for promoting blood clotting, fighting infections, and accelerating wound healing. Collectively, the results revealed that Mitragyna speciosa could be a potential candidate as a therapeutic agent for wound treatments, supporting traditional claims. • Mitragyna speciosa methanol crude extract and fractions did not exhibit toxicity on 3T3 fibroblast cells up to 50 μg/mL. • Ethyl acetate and dichloromethane fractions of Mitragyna speciosa demonstrated a more rapid cell migration. • Ethyl acetate fraction of Mitragyna speciosa could stimulate angiogenesis. [ABSTRACT FROM AUTHOR]