The objective of the present work was to analyze the cytotoxic, antimicrobial activity and the action mechanism of the major component in of the Syzygium aromaticum essential oil obtained by supercritical CO₂. In this work, gingival fibroblasts were exposed to the essential oil in different concentrations for one hour: 5 μL/ml, 7.5 μL/ml and 10 μL/ml. Culture medium was used as control. Cytotoxicity analysis was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT®) method. The susceptibility was evaluated on three microorganisms Candida albicans, Escherichia coli and Staphylococcus aureus. The statistical analyses showed significant difference in cell viability for the concentration of 10 μL/mL, as compared to the control group. As a result, the plant extract had no cytotoxicity at concentrations below 10 μL/mL in human gingival fibroblasts. The interaction mode of eugenol, the major compound and main component responsible for the biological activity of the essential oil was evaluated. The molecular docking of eugenol with important proteins of the metabolic pathway of the microorganisms C. albicans, E. coli and S. aureus were performed. The results demonstrated that the compound is capable of interacting with catalytic residues of the enzymes and forming an energetically favorable system with such proteins. The results of binding free energy obtained demonstrate this capacity. For the eugenolN-myristoyltransferase (C. albicans) system, the value of ΔG_bind was –19.01 kcal/mol, for Enoyl reductase (E. Coli) ΔG_bind was equal to –11.31 kcal/mol and for SarA (S. aureus) ΔG_bind was –13.58 kcal/mol.