Objectives/Hypothesis: Low pH, hydrogen peroxide generation, and the hyperosmolarity mechanisms of antimicrobial action are ubiquitous for all honeys. In addition, manuka honey has been shown to contain high concentrations of methylglyoxal (MGO), contributing the relatively superior antimicrobial activity of manuka honey compared to non-MGO honeys. In high concentrations, manuka honey is effective in killing Staphylococcus aureus biofilms in vitro. Lower concentrations of honey, however, are desirable for clinical use as a topical rinse in chronic rhinosinusitis in order to maximize the tolerability and practicality of the delivery technique. This study, therefore, was designed to evaluate the contribution of MGO to the biofilm-cidal activity of manuka honey, and furthermore determine whether the antibiofilm activity of low-dose honey can be augmented by the addition of exogenous MGO. Study Design: In vitro microbiology experiment. Methods: Five S. aureus strains (four clinical isolates and one reference strain) were incubated to form biofilms using a previously established in vitro dynamic peg model. First, the biofilm-cidal activities of 1) manuka honey (790 mg/kg MGO), 2) non-MGO honey supplemented with 790 mg/kg MGO, and 3) MGO-only solutions were assessed. Second, the experiment was repeated using honey solutions supplemented with sufficient MGO to achieve concentrations exceeding those seen in commercially available manuka honey preparations. Results: All honey solutions containing a MGO concentration of 0.53 mg/mL or greater demonstrated biofilm-cidal activity; equivalent activity was achieved with ≤yen;1.05 mg/mL MGO solution. Conclusions: MGO is only partially responsible for the antibiofilm activity of manuka honey. Infusion of MGO-negative honey with MGO, however, achieves similar cidality to the equivalent MGO-rich manuka honey.