Purpose This paper explores the effect of therapeutic ultrasound on the mechanical and biological properties of ligament fibroblasts. Methods and Results We assessed pulsed ultrasound doses of 1.0 and 2.0 W/cm2 at 1 MHz frequency for five days on ligament fibroblasts using a multidisciplinary approach. Atomic force microscopy showed a decrease in cell elastic modulus for both doses, but the treated cells were still viable based on flow cytometry. Finite element method analysis exhibited visible cytoskeleton displacements and decreased harmonics in treated cells. Colorimetric assay revealed increased cell proliferation, while scratch assay showed increased migration at a low dose. Enzyme-linked immunoassay detected increased collagen and fibronectin at a high dose, and immunofluorescence imaging technique visualized β-actin expression for both treatments. Conclusion Both doses of ultrasound altered the fibroblast mechanical properties due to cytoskeletal reorganization and enhanced the regenerative and remodeling stages of cell repair. Lay Summary Knee ligament injuries are a lesion of the musculoskeletal system frequently diagnosed in active and sedentary lifestyles in young and older populations. Therapeutic ultrasound is a rehabilitation strategy that may lead to the regenerative and remodeling of ligament wound healing. This research demonstrated that pulsed therapeutic ultrasound applied for 5 days reorganized the ligament fibroblasts structure to increase the cell proliferation and migration at a low dose and to increase the releasing proteins that give the stiffness of the healed ligament at a high dose. Future Works Future research should further develop and confirm that therapeutic ultrasound may improve the regenerative and remodeling stages of the ligament healing process applied in clinical trials in active and sedentary lifestyles in young and older populations. Graphical abstract