Collagen filaments derived from the two marine demosponges Ircinia oros and Sarcotragus foetidus were for the first time isolated, biochemically characterised and tested for their potential use in regenerative medicine. SDS-PAGE of isolated filaments revealed a main collagen subunit band of 130 kDa in both of the samples under study. DSC analysis on 2D membranes produced with collagenous sponge filaments showed higher thermal stability than commercial mammalian-derived collagen membranes. Dynamic mechanical and thermal analysis attested that the membranes obtained from filaments of S. foetidus were more resistant and stable at the rising temperature, compared to the ones derived from filaments of I. oros. Moreover, the former has higher stability in saline and in collagenase solutions and evident antioxidant activity. Conversely, their water binding capacity results were lower than that of membranes obtained from I. oros. Adhesion and proliferation tests using L929 fibroblasts and HaCaT keratinocytes resulted in a remarkable biocompatibility of both developed membrane models, and gene expression analysis showed an evident up-regulation of ECM-related genes. Finally, membranes from I. oros significantly increased type I collagen gene expression and its release in the culture medium. The findings here reported strongly suggest the biotechnological potential of these collagenous structures of poriferan origin as scaffolds for wound healing.