AbstractBacterial infection remains a great challenge in wound healing, especially in chronic wounds. Multidrug‐resistant organisms are increasing in acute and chronic wound infections, which compromise the chance of therapeutics. Resistance to conventional antibiotics has created an urge to study new approach/system that can effectively control wound infection and enhance healing. Wound cover/dressing must exhibit biocompatibility and effectiveness in reducing bioburden at the wound site. Collagen, a natural biopolymer, possesses advantages over synthetic and other natural materials due to its unique biological properties. It can act as an excellent wound dressing and controlled drug delivery system. Currently, antiseptic agents such as silver, iodine, and polyhexamethylene biguanide (PHMB)‐incorporated scaffolds have become widely accepted in chronic wound healing. In this study, PHMB‐incorporated collagen scaffold has been prepared and characterized using Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and differential scanning calorimetry (DSC), which showed retention of collagen nativity and integration of PHMB. The scanning electron microscopy (SEM) analysis revealed the porous structures of scaffolds. The cytotoxicity analysis showed PHMB is nontoxic at the concentration of 0.01% (wt/wt). The agar diffusion test and bacterial adhesion study demonstrated the effectiveness of PHMB‐incorporated collagen scaffold against both gram positive and negative strains. This study concludes that PHMB‐incorporated collagen scaffold could have the potential for infected wound healing.