The prominent antibacterial and quorum sensing (QS) inhibition activity of aromatic plants can be used as a novel intervention strategy for attenuating bacterial pathogenicity. In the present work, a total of 29 chemical components were identified in the essential oil (EO) of Melaleuca bracteata leaves by gas chromatography-mass spectrometry (GC-MS). The principal component was methyleugenol, followed by methyl trans-cinnamate, with relative contents of 90.46% and 4.25%, respectively. Meanwhile, the antibacterial activity and the QS inhibitory activity of M. bracteata EO were first evaluated here. Antibacterial activity assay and MIC detection against seven pathogens (Dickeya dadantii Onc5, Staphylococcus aureus ATCC25933, Pseudomonas spp., Escherichia coli ATCC25922, Serratia marcescens MG1, Pseudomonas aeruginosa PAO1 and Chromobacterium violaceum ATCC31532) demonstrated that S. aureus ATCC25933 and S. marcescens MG1 had the higher sensitivity to M. bracteata EO, while P. aeruginosa PAO1 displayed the strongest resistance to M. bracteata EO. An anti-QS (anti-quorum sensing) assay revealed that at sub-minimal inhibitory concentrations (sub-MICs), M. bracteata EO strongly interfered with the phenotype, including violacein production, biofilm biomass, and swarming motility, as well as N-hexanoyl-L-homoserine lactone (C6-HSL) production (i.e., a signaling molecule in C. violaceum ATCC31532) of C. violaceum. Detection of C6-HSL indicated that M. bracteata EO was capable of not only inhibiting C6-HSL production in C. violaceum, but also degrading the C6-HSL. Importantly, changes of exogenous C6-HSL production in C. violaceum CV026 revealed a possible interaction between M. bracteata EO and a regulatory protein (cviR). Additionally, quantitative real-time polymerase chain reaction (RT-qPCR) analysis demonstrated that the expression of QS-related genes (cviI, cviR, vioABCDE, hmsNR, lasA-B, pilE1, pilE3, and hcnB) was significantly suppressed. Conclusively, these results indicated that M. bracteata EO can act as a potential antibacterial agent and QS inhibitor (QSI) against pathogens, preventing and controlling bacterial contamination.