Honey is becoming increasingly valued for its antibacterial properties, but concrete knowledge about the mechanism of action remain incomplete. We accessed the bactericidal activity and mechanism of action of RevamilH source (RS) honey and Manuka honey, which are sources of two major honeys awarded a medical grade. The results showed that the RS honey was successful in killing Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa within a two hour period, Manuka honey had rapid activity against B subtilis. After a twenty-four hour period, both of the tested honeys had successful killed all the tested bacteria, including the methicillin resistant Staphylococcus aureus, however, Manuka honey retained activity with higher dilution, when compared to RS honey. Bee defensin-1 and H202 were the main contributing factors for the rapid bactericidal activity of RS honey. These factors weren’t found in the Manuka honey, however, it contained 44-fold higher concentrations of methylglyoxal than RS honey. Methylglyoxal was a significant bactericidal factor in the Manuka honey, but the Manuka honey still retained bactericidal activity once this was neutralised, the factors allowing this are unknown. RS and Manuka honey have distinct compositions of bactericidal factors, resulting in large differences in activity.