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The essential oil of <i>Melaleuca alternifolia</i>, commonly known as tea tree oil, has many beneficial properties due to its bioactive compounds. The aim of this research was to characterize the tea tree essential oil (TTEO) from Slovakia and its biological properties, which are specific to the chemical composition of essential oil. Gas chromatography/mass spectroscopy revealed that terpinen-4-ol was dominant with a content of 40.3%. γ-Terpinene, 1,8-cineole, and <i>p</i>-cymene were identified in contents of 11.7%, 7.0%, and 6.2%, respectively. Antioxidant activity was determined at 41.6% radical inhibition, which was equivalent to 447 μg Trolox to 1 mL sample. Antimicrobial activity was observed by the disk diffusion method against Gram-positive (G⁺), Gram-negative (G⁻) bacteria and against yeasts, where the best antimicrobial activity was against <i>Enterococcus faecalis</i> and <i>Candida albicans</i> with an inhibition zone of 10.67 mm. The minimum inhibitory concentration showed better susceptibility by G⁺ and G⁻ planktonic cells, while yeast species and biofilm-forming bacteria strains were more resistant. Antibiofilm activity was observed against <i>Pseudomonas fluorescens</i> and <i>Salmonella enterica</i> by MALDI-TOF, where degradation of the protein spectra after the addition of essential oil was obtained. Good biological properties of tea tree essential oil allow its use in the food industry or in medicine as an antioxidant and antimicrobial agent.