The use of biomaterial for tissue repair involves the interaction between materials and cells, and the coagulum formation represents the first step of tissue healing. This process is particularly critical in the oral cavity, where the wounds are immediately subjected to the masticatory mechanical stress, saliva invasion, and bacterial attack. Therefore, the present study aimed to explore the structural features and the biological activities of a hemostatic collagen sponge on human gingival fibroblasts (HGFs) and human oral osteoblasts (HOBs). The microstructure of the collagen sponge was characterized by a scanning electron microscope (SEM) and histological analysis. The porosity was also calculated. To investigate biological activities, HGFs and HOBs were cultured on the collagen sponges, and their adhesion was observed at SEM on the third day, while cell viability was investigated at the third and seventh days by Tetrazolium (MTT) assay. For osteoblasts seeded on collagen sponge the mineralization ability was also evaluated by alkaline phosphatase (ALP) assay at the seventh day, and by Alizarin red staining on the 14th. Furthermore, the gene expression of ALP and osteocalcin (OCN) was investigated after 3, 7 and 14 days. SEM images of the sponge without cells showed a highly porous 3D structure, confirmed by the measurement of porosity that was more than 90%. The samples cultured were characterized by cells uniformly distributed and adhered to the sponge surface. Proliferation ended up being promoted, as well as the mineralization ability of the osteoblasts, mainly at the mature stage. In conclusion, this collagen sponge could have a potential use for tissue healing.