The medical impact of bacterial biofilms has increased with the recognition of biofilms as a major contributor to chronic wounds such as diabetic foot ulcers, venous leg ulcers and pressure ulcers. Traditional methods of treatment have proven ineffective, therefore this article presents in vitro evidence to support the use of novel antimicrobials in the treatment of Pseudomonas aeruginosa biofilm. An in vitro biofilm model with a clinical isolate of P. aeruginosa was subjected to treatment with either lactoferrin or xylitol alone or in combination. Combined lactoferrin and xylitol treatment disrupted the structure of the P. aeruginosa biofilm and resulted in a >2log reduction in viability. In situ analysis indicated that while xylitol treatment appeared to disrupt the biofilm structure, lactoferrin treatment resulted in a greater than two-fold increase in the number of permeabilised bacterial cells. The findings presented here indicated that combined treatment with lactoferrin and xylitol significantly decreases the viability of established P. aeruginosa biofilms in vitro and that the antimicrobial mechanism of this treatment includes both biofilm structural disruption and permeablisation of bacterial membranes.