Although hydrogen peroxide (H₂O₂) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H₂O₂ accumulation in natural non-manuka honeys. H₂O₂ concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H₂O₂ producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H₂O₂ concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8±9.1 μM H₂O₂ in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9±6.8 and 495.8±9.1 μM H₂O₂, respectively. Levels of H₂O₂ increased significantly over time in both honey solutions. Contrary to this, the MGO-treated honeys generated significantly lower amounts of H₂O₂ (P<.001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H₂O₂ generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.