The effective control of pathogenic bacteria is crucial in the restoration of periodontal tissue affected by periodontitis. Guided tissue regeneration (GTR) membranes are commonly used to aid in the repair of periodontal defects. Therefore, there is a clear advantage in developing antibacterial periodontal membranes that can effectively eliminate infections and promote tissue regeneration. This study aimed to create a collagen membrane with optimal content of silver nanoparticles (AgNPs) for effective antibacterial properties and minimal toxicity to mammalian cells. Ascorbic acid-reduced AgNPs were incorporated into collagen at the ratio of 0.5%, 1%, 2%, and 3% (based on total dry weight). Collagen/AgNPs hydrogels were compressed and freeze-dried to form membranes and then were characterized. Antibacterial activity was tested against Fusobacterium nucleatum and Enterococcus faecalis, and membrane cytocompatibility was accomplished on human gingival fibroblasts. Membranes with 2% and 3% AgNPs exhibited significant antibacterial activity, while 1% showed minimal activity and 0.5% and 0% showed none. HGF cells on the 3% AgNPs membrane had poor viability, proliferation, and adhesion, but 0%, 0.5%, 1%, and 2% AgNPs membranes showed desirable cellular behavior. In conclusion, the collagen membrane with 2% AgNPs demonstrated both antibacterial capacity and excellent cytocompatibility, making it a promising choice for periodontal treatments, especially in GTR approaches.