Manuka honey, derived from the New Zealand flowering plant Leptospermum scoparium, shows promise as a topical antibacterial agent and effective chronic wound dressing. The aim of this study was to determine the non-peroxide antibacterial effects of this honey on the proteome of the common wound pathogen Staphylococcus aureus. Proteomic analysis was performed on cells treated for a short time with manuka honey compared with the proteome of untreated cells as well as cells treated with a Leptospermum honey sample without antibacterial activity. Treatment with manuka honey resulted in a significant decrease in the bacterial cell growth rate as well as downregulation of ten and upregulation of two proteins. Nine of these proteins were also differentially expressed by cells treated with the inactive Leptospermum honey, but to a lesser degree, and the rate of bacterial growth was not affected. The differentially expressed proteins have roles in ribosomal function, protein synthesis, metabolic processes and transcription. Manuka honey uniquely caused downregulation of two proteins [dihydrolipoamide dehydrogenase (DLD) and elongation factor Tu (EF-Tu)] associated with two of these pathways as well as upregulation of one stress-related protein [cold shock protein C (CspC)]. The proteomic profile following treatment with manuka honey differed from the profiles of other antibacterial agents, indicating a unique mode of action and its potential value as a novel antimicrobial agent.