Wounds with healing difficulties may lead to chronic ulcers. Gamma irradiation causes cellular damage that can delay tissue regeneration of wounds. Human amnion (HAM) is a promising wound graft because it contains many healing factors, such as growth factors and anti-inflammatory cytokines. The aim of this work is to prove the therapeutic effect of amniotic stromal layer (ASL) grafts in wounded rats post-irradiation. The impact of two different preparations of ASL grafts (fresh versus cultivated) was compared by skin grafting of two groups of wounded rats; each rat has two equivalent areas of full-thickness wound (FTW). The healing was suppressed by exposing all rats to 6 Gy gamma-irradiation. The wound on the right side was treated by skin grafting with one preparation of ASL, whereas the left-sided wound was untreated as a control. The improvement of wounds was evaluated by clinical observation, histologic examinations, and immunostaining of the expressed tumor necrosis factor-alpha (TNF-α) and epidermal growth factor (EGF). Significant differences were obtained among the different treatments concerning the contraction of wounds. The complete healing of wounds with the perfection of skin appendages was achieved only in the treated wounds with the cultivated preparation of ASL within 11 days post-wounding. In general, ASL grafts reduced the degree of inflammation, accelerated the formation of granulation tissues, reduced the expression of TNF-α and induced the expression of EGF in the treated wounds; compared to the untreated control. In conclusion, ASL is histologically compatible and promising for wound grafting and skin rejuvenation.