Objectives: The aim of our study is to look for the different Gram + and Gram - germs present in the biofilm of thermoplastic appliances and to note the qualitative modifications of the aligners’ bacterial flora after the use of the tea tree essential oil. Materials and Methods: Thirty-four patients were involved in the study. They were divided into two equal groups: a control group using a toothbrush and toothpaste as cleaning means and a test group using the tea tree essential oil as a supplement. Results: The results showed that the bacterial flora of the thermoplastic device is very diverse with a predominance of the genus Staphylococcus. It was isolated in more than half of the devices used by the control group. In terms of species, Enterobacter cloacae complex and Staphylococcus aureus were the most represented. In addition to the bacteria, usually found in the oral cavity, several other non-oral strains were frequently isolated, underscoring the importance of hand hygiene. The tea tree essential oil showed its effectiveness on Staphylococcus, which was significantly less encountered in the test group (p = 0.031). Conclusion: Thermoplastic appliances contain a diverse bacterial flora; Tea tree essential oil can be used for disinfecting purpose.