Hypothesis: Chlorhexidine (CLX) is a good antimicrobial agent, but its use in treatment of wounds is limited because of its cytotoxicity towards human fibroblasts. A delivery system, able to release CLX in a localized and prolonged manner, could guarantee antimicrobial activity with reduced cytotoxic. Thus in this work the preparation and characterization of chitosan/montmorillonite composite films containing CLX, able to offer a prolonged CLX release, is described. The antimicrobial and antibiofilm activities and cytotoxicity of films were investigated. Experimental: CLX was intercalated between the layers of montmorillonite (MONT-Na), and the intercalated product (MONT-CLX) was characterized by X-ray powder diffraction (XRPD), thermogravimetric analysis (TGA) and FT-IR spectroscopy. Then chitosan/MONT-CLX films were prepared and characterized. For comparison, films loaded with neat CLX and MONT-Na/CLX were prepared. All prepared films were tested for their antimicrobial and antibiofilm activities. Cytotoxicity towards human skin keratinocytes and human fibroblasts HuDe was evaluated as well. Findings: All prepared films showed good antimicrobial and antibiofilm activities. As concerns cytotoxicity the film containing MONT-CLX at 1% CLX concentration resulted no cytotoxic. These results confirm the potential use of chitosan films containing MONT-CLX as a potential wound dressing material to prevent microbial colonization in wounds. (C) 2016 Elsevier Inc. All rights reserved.