Adulteration of tea tree (Melaleuca alternifolia) oil with cheaper essential oils—such as eucalyptus oil—is a major threat to the Australian tea tree oil industry. Near-infrared spectroscopy may offer a rapid, cheap and non-destructive method of assessing adulteration levels in tea tree oil. In this study, near-infrared (NIR) spectra were collected through PCR tubes containing tea tree oil samples with varying concentrations of eucalyptus oil adulterant. Partial least squares regression was able to predict the level of adulteration in the tea tree oil samples with moderate accuracy (R2pred of 0.97 and RMSEP of 5.9% v/v). Furthermore, PLS-DA could discriminate between tea tree oil samples with a high level of adulteration (> 10% eucalyptus oil) and low or no adulteration (0–5% eucalyptus oil) with 93% accuracy for cross-validation and 100% accuracy in the dependent test set. This supports the use of through-container NIRS as a potential quality control method for detecting adulteration of pure tea tree oil samples.