Since the traditional methods of treatment have proven ineffective against chronic wounds involving biofilms, the present study evaluates the in vitro efficacy of a novel wound gel comprising an antibiofilm DispersinB® -enzyme and a broad-spectrum antimicrobial triclosan against chronic wound-associated bacteria. The antimicrobial and antibiofilm activity of DispersinB®-triclosan wound gel was tested against chronic wound-associated bacteria such as Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, coagulase-negative Staphylococci, Acinetobacter baumannii and Klebsiella pneumoniae using a quantitative culture method and a biofilm assay. The DispersinB®-triclosan wound gel showed more enduring antimicrobial activity against the test organisms compared to commercial wound gels Silver-Sept™ and IODOSORB. When DispersinB®-triclosan wound gel was tested against preformed biofilm, it showed 2-4 log10 reduction in the biofilm embedded bacterial cells. Furthermore, DispersinB®-triclosan wound gel compared to IODOSORB was significantly more effective in inhibiting biofilm in MRSA and A. baumannii (P < 0.05). This study indicates the potential application of a broad-spectrum DispersinB®-triclosan wound gel with both antibiofilm and antimicrobial activity in biofilm-based chronic wound management.