Chronic non-healing wounds affect a significant number of patients worldwide. Although the etiologies of these wounds are varied, bacterial infection has been suggested as a major factor responsible for the perpetual inflammation and tissue destruction observed in such wounds. Recent evidence has emerged suggesting that bacterial biofilms in particular may have a significant role in this process. At the same time, topical negative pressure dressing is gaining acceptance as a therapy which promotes healing in recalcitrant wounds. In this study an in vitro Pseudomonas aeruginosa biofilm model was developed to mimic potential surface wound biofilms. Topical negative pressure dressing was applied to the model and the effects of topical negative pressure dressing on the in vitro wound biofilms were examined using both quantitative microbiological counting technique and imaging studies. The results demonstrated a small but statistically significant reduction in biofilm bacteria at 2 weeks when exposed to topical negative pressure. When this was combined with silver impregnated foam, the reduction was far more significant and was observable within 24 hours. Microscopically, it was also noted that topical negative pressure compressed the biofilm architecture with a reduction in thickness and diffusion distance. © 2011 by the Wound Healing Society.