Aims: To develop an in vitro flat-bed perfusion biofilm model that could be used to determine the antimicrobial efficacy of topically applied treatments. Methods and Results: Pseudomonas aeruginosa and Staphylococcus aureus biofilms were grown within continuously perfused cellulose matrices. Enumeration of the biofilm density and eluate was performed at various sampling times, enabling determination of the biofilm growth rate. Two antimicrobial wound dressings were applied to the surface of mature biofilms and periodically sampled. To enable real-time imaging of biofilm growth and potential antimicrobial kinetics, a bioluminescent Ps. aeruginosa biofilm was monitored using low-light photometry. Target species produced reproducible steady-state biofilms at a density of c. 107 per biofilm support matrix, after 24-h perfusion. Test dressings elicited significant antimicrobial effects, producing differing kill kinetic profiles. There was a good correlation between photon and viable count data. Conclusions: The model enables determination of the antimicrobial profile of topically applied treatments against target species biofilms, accurately differentiating bactericidal from bacteriostatic effects. Moreover, these effects could be monitored in real time using bioluminescence. Significance and Impact of the Study: This is the first in vitro biofilm model which can assess the antimicrobial potential of topical therapies in a dynamic growth environment.