In vitro assays using a variety of essential oils revealed a particularly high antibacterial effect of Australian tea tree oil (TTO) on a great number of gram-negative and gram-positive bacteria of unrelated phylogenetic origin. In the present study, the susceptibility of cell wall-less bacteria such as the human pathogenic bacterium Mycoplasma pneumoniae to Australian tea tree oil was examined. The minimum inhibitory concentration (MIC) was determined to be 0.006% (v/v) TTO for the wild type and to 0.003% (v/v) TTO for mutants of M. pneumoniae which lost the ability to adhere to host cells (cytadherence-negative). The MIC and the MBC (minimum bactericidal concentration) for M. pneumoniae are 100 times lower than those for all other eubacteria tested. Electron microscopy with negatively stained cells as well as with ultrathin sections revealed a tendency to ovoid or round cells after oil treatment whereas the untreated cells of the wild type exhibit a flask-shaped morphology with a tip-like structure at one pole of the cell. The integrity of the mycoplasmal membrane seems not to be affected by TTO since no leakage of the Mycoplasma cell was observed after oil treatment. In the HET-CAM test TTO did not show any visible signs of irritation in concentrations less than 25%. Although the active component in TTO that has anti-mycoplasmal activity is not known, it seems very promising to use TTO tentatively for mouth washing and inhalation in case of Mycoplasma-pneumoniae-infection.