The antibacterial activity of honeys derived from the endemic flora of the southwest corner of Western Australia, including the trees Jarrah (Eucalyptus marginata) and Marri (Corymbia calophylla), remains largely unexplored. Investigation of these honeys showed minimum inhibitory concentrations (MICs) of 6.7–28.0% (w/v) against Gram positive and negative bacteria. Honey solutions showed enhanced antibacterial activity after hydrogen peroxide was allowed to accumulate prior to testing, with a mean MIC after accumulation of 14.3% compared to 17.4% before accumulation. Antibacterial activity was reduced after treatment with catalase enzyme, with a mean MIC of 29.4% with catalase compared to 15.2% without catalase. Tests investigating the role of the Gram negative outer membrane in honey susceptibility revealed increases in activity after destabilisation of the outer membrane. Honeys reduced both the formation of biofilm and the production of bacterial pigments, which are both regulated by quorum sensing. However, these reductions were closely correlated with global growth inhibition. Honey applied to existing biofilms resulted in decreased metabolic activity and minor decreases in viability. These results enhance our understanding of the mechanisms of antibacterial action of Jarrah and Marri honeys, and provide further support for the use of honey in the treatment of infected wounds.