Burn injuries can cause traumatic and debilitating physical trauma, with burn wounds prone to bacterial infection. This study examined in vitro the effectiveness of the silver nanoparticle based antimicrobial dressing, Acticoat™, in combination with a range of antimicrobial compounds against Staphylococcus aureus and Pseudomonas aeruginosa and investigated potential cytotoxic effects in multi-layered differentiated keratinocyte models. Acticoat™ with chlorhexidine was found to be highly effective against S. aureus and P. aeruginosa across a 3 day incubation period on pig skin models. MTT assays and histological staining of keratinocyte models revealed Acticoat™ had a cytotoxic effect following initial contact with the cells and cytotoxicity was exacerbated when dressings were coated with chlorhexidine and antimicrobial peptide formulations. Spectrophotometric analysis suggested that the silver nanoparticles may mobilise from the dressing as nanoclusters or silver salts, which may relate to the observed cytotoxicity. The bacterial strains used in this study showed a substantial tolerance to Acticoat™ with biofilm-like communities observed on the dressing surfaces. This could be mitigated with chlorhexidine, albeit with an increase in cytotoxicity. The clinical significance of these findings in terms of infection control and wound healing remain to be determined; the potential benefit of bactericidal activity must be balanced against cytotoxicity, and the prevalence and potential transmission of the silver tolerant phenotype must also be assessed.