Aims: To develop a simple, reproducible in vitro static diffusion method using cellulose disks and defined species to test antimicrobial efficacy of wound dressings. Methods and Results: Cellulose disks were inoculated by immersion in cell suspensions of target species Staphylococcus epidermidis, Candida albicans and Fusobacterium nucleatum. Test and control wound dressings were cut into equal sized squares (25 x 25 mm) and applied to the surface of 10-mm thick tryptone yeast extract agar on test beds. Following a 2-h equilibration period, inoculated cellulose disks were inserted (one per dressing) at the interface between dressing and agar surface and a small weight applied over each square. At various sampling times, disks were removed and surviving cells enumerated by viable counts. Disk to disk variation for microbial loading was assessed using S. epidermidis for both initial (n = 16) and standard treatment (n = 16) conditions. The coefficient of variation was low (< 5%) indicating good reproducibility for cell loading and treatment position on the test bed. Replicate assays (n = 6) using S. epidermidis and oxyzyme gels produced similar kill rates with low scatter (R-2 > 0.9) indicating good reproducibility between assays. Significant differences (P < 0.05) in kill rates were observed for different target species, types of dressing and test bed conditions (+/- blood and nutrients). Conclusions: The method is reproducible and useful in tracking the death kinetics of test species, enabling the comparison of different types of dressing. Significance and Impact of the Study: The reported method has significant advantages over established test procedures; it can be applied equally across a wide range of target species (including anaerobes and yeasts), a wide range of conditions, and different types of surface dressings, including those relying upon oxygen diffusion.