Tea tree hydrosol (TTH) is the aqueous phase of the distillate from the steam distillation process of the twigs and leaves of Melaleuca alternifolia. In this study, the chemical components of TTH were analyzed by gas chromatography–mass spectrometry (GC–MS) and gas chromatography–flame ionization detector (GC-FID). The major components were terpenoids (1.53 mg/mL), including monoterpenoids and sesquiterpenoids. The antibacterial activity of TTH was assessed by paper disc agar diffusion and broth dilution methods. Terpinen-4-ol (MIC: 0.31-0.62 mg/mL), 2- endo-hydroxy-1,8-cineole (MIC: 1.25-2.50 mg/mL) and p-menth-3-ene-1,2-diol (MIC: 0.62-2.50 mg/mL) were the main antibacterial components of TTH. The results showed that the major components of TTH and tea tree oil (TTO) had the same parent structures, but different degrees of oxidation. Terpinen-4-ol was the most abundant component in both TTH (624.2 μg/mL) and TTO (35-48%), and played a major role in antibacterial activities. This is the first report on the chemical components and antibacterial activities of TTH and their correlation with TTO, which demonstrates the potential application of TTH as a natural antimicrobial agent in health care products.