Through the use of clinical locomotor and ultrasound measurements, this study was aimed at investigating the impact of advanced platelet-rich fibrin (A-PRF), mesenchymal stem cell-derived microvesicles (MVs), and zinc oxide nanoparticles (ZnO-NPs) on the healing of tendon defects in rabbits. Thirty-six adult male New Zealand rabbits were allocated into four equal groups. An Achilles tendon defect was induced. The control group was left untreated, A-PRF group treated with application of PRF membrane, ZnO-NP group was treated with 0.2% ZnO-NPs, and MV group treated with MSC-derived MVs. Healing was assessed through clinical and ultrasound evaluations at 2-, 4- and 6-weeks. Results revealed that all the treatment groups had better outcomes than the control group regarding lameness, pain during complete flexion of the tarsal joint, adhesion, and inflammation. The MVs and ZnO-NPs significantly decreased lameness and adhesion than the A-PRF and control groups. Ultrasonographic examination revealed a normal echogenic fibrillar pattern with normal margins and no evidence of ultrasonographic abnormalities in the MVs or ZnO-NP groups compared to other groups. Overall, ultrasonographic locomotive observation revealed the MVs and ZnO-NP groups' superiority in treating Achilles tendon defects compared with the A-PRF and control groups when used to fill a tendon defect in a rabbit model.