Injury and degeneration of tendons can be highly debilitating and can result in substantial pain, disability, and health-care costs. Nano-sized fibers have a much wider surface area than conventionally produced fibers, which can hold composite materials more compactly and thus provide greater mechanical capabilities. In this study, the injured tendon was treated by electrospun PVA mats containing Eucalyptus extract and histopathological results of healing were evaluated. 45 male Wistar rats were used in this study. For this study, 45 male Wistar rats were prepared and a partial thickness tenotomy was created on right hindlimbs. All rats were divided into three groups (n = 15) and three sub-groups (n = 5) including, Eucalyptus extract-loaded nanofibers, PVA nanofiber, and without any treatment as a control group. Histological samples were taken on days 14, 28, and 42. The histological analysis on day 14 indicated no significant difference was observed between all groups (p > 0.05). While on days 28 and 42 post-rupture indicated a higher regenerating activity and capacity in the Eucalyptus extract-loaded nanofibers than PVA nanofiber and control groups (p ≤ 0.05). In summary, these results suggest that the Eucalyptus extract-loaded nanofibers mats promoted the healing process of damaged Achilles tendon in rats.