Infected wounds are global healthcare problems since they can lead to dysfunctioning in organs and even result in death in severe cases. In this study, antibacterial wound dressings were prepared to prevent possible infection at the damaged host region. For this purpose, three natural polymers namely heparin, chitosan and alginate were used as anionic, cationic and sulfated polysaccharides, respectively, due to their versatile properties as being nontoxic, biocompatible, biodegradable and antibacterial. Heparin/chitosan/alginate based complex polyelectrolyte scaffolds containing different amounts of heparin were prepared by freeze drying technique. The effects of calcium chloride, which is used as crosslinker for alginate, on the stability of scaffolds were tested. Presence of functional groups and morphological structures of the scaffolds were examined in detail by using FTIR, XPS and SEM. Antibacterial activities against Staphylococcus epidermidis (S. epidermidis) were investigated under in vitro conditions. Presence and the amount of heparin in the scaffolds significantly enhanced the antibacterial activities. The results demonstrated that heparin/chitosan/alginate polyelectrolyte scaffolds have very high potential to be used as therapeutic wound dressings.