Background and Objective: Bacterial contamination and consecutive infections had been highly concerned. Contamination has increasingly become prevalent which occurred in food, processing-food factories and laboratories resulting in severe consequences, more importantly, many dangerous diseases for humans. Thus, the investigation of natural compounds which are non-antibiotics, could inhibit the growth of pathogens and restrict the side effects to apply in disinfections as well as preventing bacterial contamination is imperative. The intention of this research was to study the antimicrobial activity of Melaleuca essential oils (consisting of Melaleuca alternifolia and Melaleuca citrina oils) which was also called tea tree oil (TTO), grounded on the chemical components. Materials and Methods: The bioactivity of TTO was evaluated by the combination of several methods including agar well diffusion and broth micro-dilution to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The compositions of Melaleuca essential oils were identified by GC-MS analysis. Results: There were sixteen and twelve components obtained in M. alternifolia and M. citrina oils. The main compounds of M. alternifolia and M. citrina were eucalyptol (51.88 and 76.55%) and terpene-4-ol (30.08 and 4.93%), respectively. The Melaleuca oils showed high antibacterial activity which was assessed with two bacterial species representing Gram-positive and Gram-negative bacteria, Listeria monocytogenes and Escherichia coli. The antimicrobial efficacies were recorded at high values in L. monocytogenes with inhibition zones (13.67 and 16.00 mm), MICs were 0.2 and 1.25%, E. coli with inhibition zones (12.33 and 12.67 mm), MICs were 0.3 and 3.0% for M. alternifolia and M. citrina, respectively. Conclusion: The bioactivity of Melaleuca essential oils was proven that be effective in resisting both Gram-positive and Gram-negative bacteria. Thus, the TTO could be examined as a potential selection substitution for antibiotics.