BackgroundThe amniotic membrane (AM) has shown immense potential in repairing wounds due to its great regenerative qualities. Although the role of AM as a biological scaffold in repairing wounds has been studied well, the tissue regenerative potential of AM‐derived mesenchymal stem cells (MSCs) and conditioned media (CM) derived from it remains to be discovered as of now. Here, we examined the wound healing abilities of fresh and frozen thawed rabbit AM (rAM) along with the MSCs and their lyophilised CM in rabbits challenged with skin wounds.MethodsTo elucidate the role of rAM‐MSCs and its CM in repairing the wound, we isolated it from the freshly derived placenta and characterised their differentiation potential by performing an in vitro tri‐lineage differentiation assay besides other standard confirmations. We compared the wound repair capacities of rAM‐MSCs and lyophilised CM with the fresh and cryopreserved AM at different timelines by applying them to excision wounds created in rabbits.ResultsBy monitoring wound contractions and tissue histology of wounded skin at different time points after the application, we observed that rAM‐MSCs and rAM‐MSC‐derived CM significantly promoted wound closure compared to the control group. We also observed that the wound closure capacity of rAM‐MSCs and rAM‐MSC‐derived CM is as efficient as fresh and cryopreserved rAM.ConclusionOur findings suggest that rAM‐MSCs and rAM‐MSC derived CM can be effectively used to treat skin wounds in animals and correctly delivered to the damaged tissue using AM as a bioscaffold, either fresh or frozen.