Aim The aim of the study was to evaluate the ability of in vitro planktonic and immobilized cell models for determining the antimicrobial efficacy of common antimicrobial wound dressings. Methods and Results Five strains of Acinetobacter baumannii, Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus were tested against four antimicrobial wound dressings containing silver, honey or polyhexamethylene biguanide (PHMB), using both a planktonic and immobilized cell model. Across all species and models used, the nanocrystalline silver coated dressing demonstrated the best antimicrobial activity being as good if not better than all the other dressings. The planktonic cell model was less effective at differentiating the dressings on antimicrobial performance as the immobilized cell model indicating that a diffusion barrier had a significant impact on the performance of some dressings. In the presence of the diffusion barrier, antimicrobial impact of the Honey and PHMB dressings was significantly reduced, particularly in the case of A. baumannii. Activity was at least an order of magnitude lower in the immobilized cell model vs the planktonic cell model. Conclusions The use of a planktonic cell model within standard tests may overestimate the efficacy of honey and PHMB. The use of an immobilized cell model provides a more demanding test for antimicrobial dressings allowing dressing to dressing and pathogen to pathogen differences to be more clearly quantified. Significance and Impact of the Study The introduction of planktonic and immobilized cell models as part of testing regimens for wound dressings will provide a more thorough understanding of their antimicrobial and anti-biofilm properties.