Background: Essential oils are odorous, volatile products of plant secondary metabolism, which are found in many leaves and stems. They show important biological activities, which account for the development of aromatherapy used in complementary and alternative medicine. The essential oil extracted from Melaleuca quin-quenervia (Cav.) S.T. Blake (paperbark) (MQ-EO) has various functional properties. Purpose: The aim of this study is to investigate the chemical composition of MQ-EO by using gas chromatography-mass spectrometry (GC-MS) and evaluate its tyrosinase inhibitory activity. Methods: Gas chromatography-mass spectrometry (GC-MS)-based metabolomics was used to identify 18 components in MQ-EO. The main components identified were 1,8-cineole (21.60%), alpha-pinene (15.93%), viridiflorol (14.55%), and alpha-terpineol (13.73%). B16 melanoma cells were treated with alpha-melanocyte-stimulating hormone (alpha-MSH) in the presence of various concentrations of MQ-EO or its major compounds. Cell viability was accessed by MTT assay and cellular tyrosinase activity and melanin content were determined by using spectro-photographic methods. The antioxidant mechanism of MQ-EO in alpha-MSH stimulated B16 cells was also investigated. Results: In alpha-melanocyte-stimulating hormone (alpha-MSH)-stimulated murine B16 melanoma cells, MQ-EO, 1,8cineole, alpha-pinene, and alpha-terpineol significantly reduced melanin content and tyrosinase activity. Moreover, MQEO, 1,8-cineole, alpha-pinene, and alpha-terpineol decreased malondialdehyde (MDA) levels. In addition, restored glutathione (GSH) levels, glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activities were increased in alpha-MSH-stimulated B16 cells. MQ-EO not only decreased apoptosis but also reduced DNA damage in alpha-MSH stimulated B16 cells. These results showed that MQ-EO and its main components, 1,8-cineole, alpha-pinene, and alpha-terpineol, possessed potent anti-tyrosinase and anti-melanogenic activities besides the antioxidant properties. Conclusions: The active functional components of MQ-EO were found to be 1,8-cineole, alpha-pinene, and alpha-terpineol. Consequently, the results of present study suggest that MQ-EO is non-cytotoxic and can be used as a skin-whitening agent, both medically and cosmetically.