Decellularization, preservation protocol and storage time influence the biomechanical and biological properties of allografts and xenografts. Here, we examined the consequences of storage time on the antibacterial, angiogenic and biocompatibility properties of the decellularized placental sponge (DPS) in vitro and in vivo. The DPS samples were preserved for one, three and six months at −20 °C. The decellularized scaffolds showed uniform morphology with interconnected pores compared with not decellularized sponges. Storage time did not interfere with collagen and vascular endothelial growth factor contents, and cytobiocompatibility for Hu02 fibroblast cells. Chorioallantoic membrane assay and subcutaneous implantation indicated a decreased new vessel formation and neovascularization in six months DPS sample compared with other experimental groups. The number of CD4+ and CD68+ cells infiltrated into the six months DPS on the implanted site showed a significant increase compared with one and three months sponges. The antibacterial activities and angiogenic properties of the DPS decreased over storage time. Three months preservation at −20 °C is suggested as the optimal storage period to retain its antibacterial activity and high stimulation of new vessel formation. This storage protocol could be considered for preservation of similar decellularized placenta-derived products with the aim of retaining their biological properties.